Bio-Dot® Microfiltration Apparatus Instruction Manual - Bio-Rad
Contents
1. Bio Dot Microfiltration Apparatus Instruction Manual Catalog Numbers 170 6545 170 6547 For technical service call your local Bio Rad office or in the U S call 1 800 424 6723 Section 1 1 1 Section 2 2 1 2 2 Section 3 3 1 3 2 Section 4 4 1 4 2 Section 5 Section 6 6 1 6 2 Section 7 7 1 7 2 7 3 7 4 Section 8 8 1 8 2 Section 9 Section 10 Section 11 12 1 12 2 Section 12 Table of Contents Page Introd CHOn ien NEEE a xi E a a 1 SPECHICALIONS eR 1 Special Handling Features essesssssuuss 2 AULOCIAVING mm 2 Chemical Stablility 37 ci in ere t es 2 Bio Dot Assembly 5 4 ona e coppa ga RNU RIDES Ur nra E LEA AN KRN E OVERKA KUNA NE 3 CI ae p 3 Hel ptul IMS sass iter tte ree lam RE ERO ea 5 Protein Blotting 1 eorr eer or rione ener etri eno Deie 7 Immunoassay Procedure eener oii oeenn eE AEAEE A EEEE EARRAN 7 Special Protein Blot Applications sssssssssseeee 9 DNA Blottiiig 0051 1e ree Cu ru iere o e Saunu EA 10 RNA Blotting ii i a erre rre enano evan einen Eee naa an FRA Re ana nain 11 Alkaline RNA Denaturation and Fixation eeeenene 11 Glyoxal RNA Denaturation and Fixation eese 11 Hybridization Protocols for Nucleic Acids 13 Probe Recommendations eessssseseeeeeeeeneeee nennen 12. L O0utlet port O O 4 2 2 Diagonal crossing pattern for tightening screws in the Bio Dot apparatus Attach a vacuum source house vacuum or a vacuum pump to the flow valve with a waste trap set up and positioned between the vacuum outlet and the flow valve Turn on the vacuum and set the 3 way valve to apply vacuum to the apparatus flow valve setting 1 Figure 3 With vacuum applied repeat the tightening process using the diagonal crossing pattern Tightening while vacuum is applied ensures a tight seal preventing cross contamination between slots Failure to tighten screws during application of vacuum prior to starting the assay may lead to leaking between the wells Adjust the flow valve so that the vacuum manifold is open to air pressure flow valve setting 2 Figure 3 Apply 100 ul buffer to all 96 sample wells Use of the 8 channel pipet and buffer reservoirs see Section 13 for ordering information will simplify the process of adding solutions to the Bio Dot apparatus Addition of buffer is necessary to rehydrate the membrane following the vacuum procedure in step 7 If this step is not performed prior to applying samples assay results will show halos or weak detection signal Gently remove the buffer from the wells by vacuum flow valve setting 3 Figure 3 Watch the sample wells As soon as the buffer solution drains from all the wells adjust the flow valve so that the unit is exposed to air and disconnect the va
3. 1L 20 mM Tris pH 7 5 500 mM NaCI 0 05 Tween 20 Add 0 5 ml Tween 20 to 1 L of TBS Blocking Solution 100 ml 1 BSA TBS Add 1 0 g bovine serum albumin BSA to 100 ml TBS Stir to dissolve Antibody Buffer 200 ml 1 BSA TTBS Add 2 g BSA to 200 ml TTBS Stir to dissolve 100 ml should be reserved for primary antibody and an equal volume for dilution of the secondary antibody conjugate Primary Antibody Solution 100 ml Dilute antigen specific primary antibody to the appropriate titer in 100 ml of antibody buffer Secondary Antibody Solution 100 ml Dilute species specific Bio Rad secondary antibody conjugate 1 3 000 by adding 33 ul of conjugate to 100 ml of antibody buffer Color Development Solution The specific chemicals and buffers are dependent on the enzyme conjugate being used See the Immun Blot assay kit instruction manual for details on how to make the appropriate solution 17 8 2 Solutions for Zeta Probe Membrane for Protein Applications When immobilizing antigen onto the Zeta Probe membrane the immunoassay must be performed in a separate container following removal of the membrane from the Bio Dot apparatus Two methods of blocking are given Method A uses nonfat dry milk Jerome and Jiehning 1986 Johnson et al 1984 as the blocking agent Method B uses gelatin and 1 methyl 2 pyrrolidinone MPO as the blocking agents The solutions for the two methods are not interchangeable If Method A is chosen all
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5. Comp es em em amy a gt Cem Ca 2 em am am eto Fig 1 Diagram of proper Bio Dot apparatus assembly 4 Fig Always use forceps or wear gloves when handling membranes Prewet the nitrocellulose or Zeta Probe membrane by slowly sliding it at a 45 angle into wetting solution Note PVDF membrane is not recommended Wet nitrocellulose in 6x sodium sodium citrate SSC for nucleic acid applications and in tris buffered saline TBS for protein blotting Wet Zeta Probe membrane in distilled water See Sections 9 and 10 for solution preparation A 10 minute soak is recommended for complete wetting of the membrane to insure proper drainage of solutions Remove the membrane from the wetting solution Let the excess liquid drain from the membrane Touching the membrane to a sheet of filter paper is a simple method for removing excess buffer Lay the membrane on the gasket in the apparatus so that it covers all of the holes The membrane should not extend beyond the edge of the gasket after the Bio Dot apparatus is assembled Remove any air bubbles trapped between the membrane and the gasket Note PVDF membrane is not recommended Place the sample template on top of the membrane The guide pins ensure that the template will be properly aligned Finger tighten the four screws When tightening the Screws use a diagonal crossing pattern to ensure uniform application of pressure on the membrane surface see Figure 2 O O b d
6. are to be done on the membrane then the membrane can be dried When reprobing do not allow the membrane to dry between hybridizations Expose moist membranes between plastic wrap or enclosed in a sealable plastic bag Do not allow a wet membrane to come in contact with the film because a wet Zeta Probe membrane will stick to the film and moisture on the film can cause artifacts Note To increase the rate of hybridization include 10 dextran sulfate final concentration in the hybridization solution Maniatis 1982 Prewarm hybridization solution to 50 C Denature the probe and carrier as above Special care must be taken to ensure uniform mixing of the denatured probe with the hybridization solution since the solution is quite viscous at 50 C 7 4 Probe Stripping and Rehybridization If reprobing is desired do not allow the membrane to dry between hybridizations The membrane should be stripped as soon as possible after autoradiography 1 Wash two times 20 minutes each in a large volume of 0 1x SSC 0 5 SDS at 95 C 2 Check membrane for removal of autoradiography patterns by overnight exposure 16 Section 8 Solutions for Protein Applications 8 1 Solutions for Nitrocellulose Membrane Tris Buffered Saline 1x TBS 2L 20 mM Tris HCl pH 7 5 500 mM NaCl Dissolve 4 84 g Tris 58 48 g NaCl in 1 5 L distilled deionized H O Adjust to pH 7 5 with HCI Adjust the volume to 2 L with ddH O Tween 20 Wash Solution 1x TTBS
7. is ready for autoradiography If no further cycles of hybridization are to be done on the membrane the membrane can be dried When reprobing do not allow the membrane to dry between hybridizations Make the autoradiographic exposure with the moist membrane wrapped in plastic wrap Do not allow the wet membrane to come in contact with the film because a wet Zeta Probe membrane will stick to the film and any moisture on the film will cause artifacts black spots 14 7 3 Hybridization Protocols for RNA Probes The following protocols are for RNA probes to DNA blots Casey and Davidson 1977 contains protocols for RNA RNA hybridizations Prehybridization 1 Place the blotted membrane inside a heat sealable plastic bag Seal three sides leaving the top side open Pipet in the prehybridization solution For DNA Bound to For DNA Bound to Zeta Probe Membrane Nitrocellulose Bio Rad Laboratories 1987 Jerome and Jaehning 1986 50 formamide 50 formamide 1 5x sodium sodium phosphate EDTA SSPE 0 1 SDS 1 SDS 5x SSPE 0 5 non fat dry milk 5x Denhardt s solution 200 ug ml carrier RNA 200 ug ml denatured salmon sperm DNA 500 ug ml denatured salmon sperm DNA The DNA must be denatured before adding it to the prehybridization solution by heating at 100 C for 5 minutes followed by rapid cooling in ice 3 Seal the bag and incubate DNA Bound to DNA Bound to Zeta Probe Membrane Nitrocellulose 30 minutes at 50 C 4 ho
8. on the side of the wells Rinse with 500 ul cold 10 mM NaOH 1 mM EDTA Apply vacuum flow valve setting 1 Figure 3 until the sample wells are dry Disassemble the Bio Dot apparatus Remove the blotted membrane and rinse it in 2x SSC 0 1 sodium dodecyl sulfate SDS Nitrocellulose membranes must be baked under vacuum for 2 hours at 80 before hybridization The Zeta Probe membrane is ready for hybridization If hybridization is not to be undertaken within 2 days then bake the Zeta Probe membrane under vacuum for 30 minutes at 80 C The Zeta Probe membrane and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C 6 2 Glyoxal RNA Denaturation and Fixation 1 2 3 Prepare RNA samples to the following final concentrations 50 dimethyl sulfoxide DMSO 10 mM NaH PO pH 7 0 1 M glyoxal Incubate the RNA for 1 hour at 50 C Cool the samples on ice Always wear gloves when handling blotting membranes Prewet the blotting membrane by placing it gently at a 45 angle into a tray of wetting solution The Zeta Probe membrane is wetted in distilled water nitrocellulose is wetted in 6x SSC see Section 10 for solution preparation 11 Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Remember to apply the vacuum and then retighten the screws that hold the apparatus together Samples and wash solutions may be applied with a standard pipet or a Cost
9. ratio during hybridization For hybridization on Zeta Probe membrane the following is recommended Probe specific activity 10 cpm ug probe Probe concentration in the hybridization mixture 108 counts ml 10 50 mg ml Probe length 200 1 000 bp Optimal probe specific activity and concentration can vary according to available hybridization sites and exposure time Alternative hybridization protocols are necessary when probe lengths vary outside this recommended range Bio Rad Laboratories 1987 Probe cleanup is essential to minimize background Unincorporated nucleotides present after probe preparation contribute to hybridization background The most effective cleanup method is by column chromatography This can be done quickly and easily with the Bio Spin chromatography columns Bio Spin 6 columns catalog number 732 6000 or Bio Spin 30 columns catalog number 732 6004 After cleanup denature double stranded probes by heating to 95 100 C for 5 minutes Then cool rapidly on ice Use the probe as soon as possible after preparation There are several hybridization protocols given in this section All protocols are for using DNA probes to hybridize to either DNA or RNA The 7 SDS hybridization protocol requires minimal prehybridization treatment and has a high signal strength and low background Further references and techniques for hybridizing to the Zeta Probe membrane may be found in the Zeta Probe membrane instruction manual
10. solutions must be preparied according to Method A if Method B is chosen all solutions must be prepared according to Method B TBS Tris Buffered saline 2 L Same as nitrocellulose membrane solution TTBS Tween 20 Tris buffered saline 2 L Method A Add 3 ml Tween 20 to 1 L of TBS This solution is used when nonfat dry milk is the blocking agent OR Method B Add 2 ml Tween 20 and 50 ml MPO to 1 L of TBS This solution is used when gelatin and MPO are the blocking agents Blocking solution 100 ml Method A Add 5 g of nonfat dry milk to 100 ml of TBS OR Method B Add 3 g of gelatin to 100 ml of TBS Warm to 37 C to dissolve the gelatin with stirring Cool before use Antibody buffer 200 ml Method A Add 10 g of nonfat dry milk to 200 ml TTBS 100 ml is used for the primary antibody solution and 100 ml is used for conjugate dilution OR Method B Add 2 g gelatin to 200 ml TTBS that already contains 5 MPO Warm to 37 C to dissolve gelatin and cool before adding antibody 100 ml is used for the primary antibody solution and 100 ml is used for conjugate dilution Primary antibody solution 100 ml Same as nitrocellulose membrane antibody buffer solution Secondary antibody solution 100 ml Same as nitrocellulose membrane antibody buffer solution Color development solution The specific chemicals and buffers are dependent on the enzyme conjugate being used See the Bio Rad Immun Blot assay kit instruction manual
11. viral DNA Proc Natl Acad Sci USA 77 6851 6855 1980 Bresser J and Gillespie D Quantitative binding of covalently closed circular DNA to nitrocellulose in Nal Anal Biochem 129 357 364 1983 Casey J and Davidson N Rates of formation and thermal stabilities of RNA DNA and DNA DNA duplexes at high concentrations of formamide Nucleic Acids Res 4 1539 1552 1977 Chen CW and Thomas CA Jr Recovery of DNA segments from agarose gels Anal Biochem 101 339 341 1980 Cleveland PH et al Rapid and efficient immobilization of soluble and small particulate antigens for solid phase radioimmunoassays J Immunoassay 2 117 136 1981 Cunningham M Spot blot a hybridization assay for specific DNA sequences in multiple samples Anal Biochem 128 415 421 1983 Faulstich H et al Alpha and beta galactosidases bound to nylon nets FEBS Lett 48 226 229 1974 Gershoni JM and Palade GE Protein blotting principles and applications Anal Biochem 131 1 15 1983 Harpold MM et al Construction and identification by positive hybridization translation of a bacterial plasmid containing a rat growth hormone structural gene sequence Nucleic Acids Res 5 2039 2053 1978 Hawkes R et al A dot immunobinding assay for monoclonal and other antibodies Anal Biochem 119 142 147 1982 Herbrink P et al The antigen spot test AST a highly sensitive assay for the detection of antibodies J Immunol Methods 48 293 298 1982 Holland LJ
12. 3 Hybridization Protocols for DNA or RNA Bound to Nitrocellulose or Zeta Probe Membrane eranan a e aee eren nennen nnns 13 Hybridization Protocols for RNA Probes see 15 Probe Stripping and Rehybridization seeeee 15 Solutions for Protein Applications 17 Solutions for Nitrocellulose Membrane eene 17 Solutions for Zeta Probe Membrane for Protein Applications 18 Solutions for Nucleic Acid Applications 19 Troubleshooting Guide eeeseeeeeeeeeeeeneee 21 Applications and References eeeeeseeeseeeeeee 24 Common Applications siena aana a a a aa nmn 24 References eoo ce A a ais a E coude aede uut 22 Ordering Information iic rare cO ripa in Ezio Der rni in Ee ri ori s 27 Section 1 Introduction The Bio Dot microfiltration apparatus can be used for any application requiring rapid immobilization and screening of unfractionated or purified proteins nucleic acids or macromolecular complexes on membranes such as nitrocellulose or Zeta Probe membrane The Bio Dot apparatus is provided as a complete unit or as a modular addition to the Bio Dot SF slot format microfiltration apparatus Conversion of the Bio Dot apparatus to the Bio Dot SF apparatus is accomplished by purchasing the Bio Dot SF module which provides the 48 we
13. 983 Viral DNA sequence detection Berg et al 1986 Brandsma and Miller 1980 Hybridization selection Harpold et al 1978 Ricciardi et al 1979 Gene product and clone selection Kranz and Gennis 1982 Plasmid analysis Bresser and Gillespie 1983 CsCl gel column or sucrose gradient fraction Cunningham 1983 Palfree and Elliott 1982 Shen et al 1980 DNA filter binding assays including DNA drug DNA protein virus host DNA synthesis etc Bresser and Gillespie 1983 DNA RNA purification Chen and Thomas 1980 Holland and Wangh 1983 Kutateladze et al 1979 Winberg and Hammarskjold 1980 12 2 References Achberger EC and Whiteley HR The role of the delta peptide of the Bacillus subtilis RNA polymerase in promoter selection J Biol Chem 256 7424 7432 1981 Allen JD and Parsons SM Nitrocellulose filter binding quantitation of the histidyl tRNA ATP phosphoribosyltransferase complex Anal Biochem 92 22 30 1979 Bennett FC and Yeoman LC An improved procedure for the dot immunobinding analysis of hybridoma supernatants J Immunol Methods 61 201 207 1983 Berg LJ et al Complementation of a bovine papilloma virus low copy number mutant evidence for a temporal requirement of the complementing gene Mol Cell Biol 6 859 869 1986 24 Bio Rad Laboratories Zeta Probe Instruction Manual Brandsma J and Miller G Nucleic acid spot hybridization rapid quantitative screening of lymphoid cell lines for Epstein Barr
14. The final volume of hybridization solution is important in reducing background For prehybridization and hybridization use 150 ul solution cm of membrane For washes use at least 350 ul cm of membrane 7 2 Hybridization Protocols for DNA or RNA bound to Nitrocellulose or Zeta Probe Membrane Prehybridization 1 Place the blotted membrane inside a heat sealable plastic bag Seal three sides leaving the top side open 2 Pipet in the correct prehybridization solution for application For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose Bio Rad Laboratories 1987 Maniatis et al 1982 Thomas 1980 1 mM EDTA 6x SSC 5096 formamide 7 SDS 0 5 SDS 5x SSC 0 5 M NaHPO pH 7 2 5x Denhardt s solution 1x Denhardt s solution 100 ug ml denatured 50 mM NaHPO pH 6 5 salmon sperm DNA 250 ug ml denatured 1mM EDTA salmon sperm DNA 13 The carrier DNA used with nitrocellulose must be denatured before adding it to the prehybridization solution Heat the DNA at 100 C for 5 minutes and cool rapidly 3 Seal the top of the bag and incubate For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 5 minutes at 65 C 2 4 hours at 68 C 8 20 hours at 42 C Hybridization 1 Cut one corner of the plastic bag Remove the prehybridization solution and replace it with a fresh batch of the same solution except when binding RNA to nitrocellul
15. age will occur during filtration applications The best method for removing the blotted membrane from the BioDot apparatus is to leave the vacuum on following the wash step With the vacuum on loosen the screws and remove the sample template Next turn off the vacuum and remove the membrane A method for applying gentle vacuum to the apparatus is to adjust the flow valve so that it is open to air the vacuum source and the vacuum manifold while the vacuum is on Then use a finger to cover the valve port exposed to the atmosphere The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve For applications using glass membranes that might break under vacuum pressure an extra piece of tubing can be attached to the flow valve to increase hydrostatic pressure during wash steps This tubing should extend approximately 2 3 feet below the level of the apparatus usually to a waste receptacle on the floor With this increased hydrostatic pressure fluid will drain from the apparatus in 3 4 minutes This type of gentle pressure is also useful for binding nucleic acids to nitrocellulose or Zeta Probe membranes Section 4 Protein Blotting 4 1 Immunoassay Procedure Detailed instructions including a comprehensive troubleshooting guide for performing immunoassays are included in the Immun Blot instruction manuals 1 Assemble the Bio Dot apparatus as described in Section 3 Prewet the membra
16. and Wangh LJ Efficient recovery of functionally intact mRNA from agarose gels via transfer to an ion exchange membrane Nucleic Acids Res 11 3283 3300 1983 Horejsi V and Hilgert Nitrocellulose membrane as an antigen or antibody carrier for screening hybridoma cultures J Immunol Methods 62 325 329 1983 Huet J et al Spot immunodetection of conserved determinants in eukaryotic RNA polymerases Study with antibodies to yeast RNA polymerases subunits J Biol Chem 257 2613 2618 1982 Jantzen K et al The DNase sensitive domain of the chicken lysozyme gene spans 24 kb Nucleic Acids Res 14 6085 6099 1986 Jerome JF and Jaehning JA mRNA transcription in nuclei isolated from Saccharomyces cerevisiae Mol Cell Biol 6 1633 1639 1986 Johnson DA et al Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose Gene Anal Tech 1 3 8 1984 Kaftos FC et al Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure Nucleic Acids Res 7 1541 1552 1979 Kane CM et al Tissue specific and species specific monoclonal antibodies to avian red cell nuclear proteins Proc Natl Acad Sci USA 79 6265 6269 1982 Karagyozov LK and Hadjiolov AA Isolation of active transcription complexes from animal cell nuclei by nitrocellulose binding J Biochem Biophys Methods 5 329 339 1982 Kranz RG and Gennis RB A quantitative ra
17. ar Octapette pipet Apply the denatured RNA and pull the sample through by passive filtration or by applying a gentle vacuum Note a method for applying gentle vacuum to the apparatus is to adjust the flow rate valve to setting 3 Use a finger to cover the valve port exposed to air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valves Rinse all wells to wash through any sample on the side of the wells Rinse with 500 ul TE Apply vacuum flow valve setting 1 Figure 3 until the sample wells are dry Disassemble the Bio Dot apparatus Remove the blotted membrane Remove glyoxal adducts by pouring 20 mM Tris HCl pH 8 0 1 mM EDTA heated to 95 C onto the membrane and agitating at room temperature until the solution cools Place the membrane in a vacuum oven at 80 C for 1 hour for the Zeta Probe membrane 2 hours for nitrocellulose Nitrocellulose membrane must be baked under vacuum for 2 hours at 80 C before hybridization If hybridization is not to be undertaken within 2 days then bake the Zeta Probe membrane under vacuum for 30 minutes at 80 C The Zeta probe membrane and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C 12 Section 7 Hybridization Protocols for Nucleic Acids 7 1 Probe Recommendations The specific activity concentration size range and purity of the probe all have an important effect on signal to noise
18. before hybridization The Zeta Probe membrane and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C 10 Section 6 RNA Blotting RNA must be denatured prior to application to Zeta Probe or nitrocellulose membranes to ensure optimal hybridization Two protocols are presented for denaturing RNA samples 6 1 Alkaline RNA Denaturation and Fixation 1 Always wear gloves when handling blotting membranes Pre wet the blotting membrane by placing it gently at a 45 angle into a tray of wetting solution The Zeta Probe membrane is wetted in distilled water nitrocellulose is wetted in 6x SSC see Section 9 for solution preparation Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Remember to apply the vacuum and then retighten the screws that hold the apparatus together Immediately before use dissolve RNA samples in 500 ul of ice cold 10 mM NaOH 1 mM EDTA Samples and wash solutions may be applied with a standard pipet or a Costar Octapette pipet Apply the denatured RNA and pull the sample through by passive filtration or by applying a gentle vacuum Note A method for applying gentle vacuum to the apparatus is to adjust the flow valve to setting 3 Use a finger to cover the valve port exposed to air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve Rinse all wells to wash through any sample
19. brane by attaching tubing to the flow valve to increase the hydrostatic pressure see Section 3 2 Perform three washes with TBS buffer using tubing rather than vacuum to speed the flow rate d Perform antibody incubations as described in Section 4 1 for protein immunoassays Section 5 DNA Blotting This section gives protocols for DNA blotting The alkaline blotting method using Zeta Probe membrane and the standard method for DNA blotting to nitrocellulose is described 1 The target DNA must be denatured prior to application to the membrane When using the Zeta Probe membrane denature the DNA sample by addition of NaOH and EDTA solution to final concentrations of 0 4 M NaOH 10 mM EDTA Heat the sample to 100 C for 10 minutes to ensure complete denaturation When applying DNA to A nitrocellulose membrane denature the DNA in the same manner The DNA must then be neutralized by adding an equal volume of cold 2 M ammonium acetate pH 7 0 to the target DNA solution Pre wet the membrane by placing the membrane gently at a 45 angle into a tray of the wetting solution Always wear gloves when handling blotting membranes Nitrocellulose membranes should be wetted in 6x SSC Zeta Probe membranes should be wetted in distilled water see Section 10 for recipes Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Apply the vacuum and then retighten the screws that hold the apparatus together Rehydrate the membran
20. cuum At this point the unit is ready for sample application Flow valve setting 1 The vacuum manifold is exposed to the vacuum source only Use for applying vacuum to the Bio Dot apparatus Bio Dot Vacuum Air Flow valve Setting 2 Bio Dot The manifold is exposed to air Use for gravity filtration procedures Vacuum Air Flow valve Setting 3 The manifold is exposed to both air and the vacuum Use this setting for gentle Bio Dot Vacuum vacuum applications where the amount of vacuum is regulated by putting a finger over the port exposed to the atmosphere Fig 3 Optional settings for the 3 way flow valve to obtain optimal performance from the Bio Dot apparatus 3 2 Helpful Hints 1 During the assay do not leave the vacuum on This may dehydrate the membrane and may cause halos around the wells Apply vacuum only until solutions are removed from the sample wells then adjust the flow valve so that the unit is exposed to air and disconnect the vacuum If some sample wells are not used in a particular assay those wells must be closed off to ensure proper vacuum to the wells in use There are three ways to close off unused wells One is to apply a 3 gelatin solution to those wells Gelatin will clog the membrane and cut off the vacuum flow to the clogged wells The second method is to cover the unused portion of the apparatus with tape to prevent air from moving through those wells The third method is to add buffer to the
21. dioimmunological screening method for specific gene products Anal Biochem 127 247 257 1982 Kutateladze TV et al New procedure of high voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids Anal Biochem 100 129 135 1979 Lin S and Riggs AD The general affinity of lac repressor for E coli DNA implications for gene regulation in procaryotes and eukaryotes 4 107 111 1975 25 Locker D Motta G Detection of antibody secreting hybridomas with diazobenzyloxymethyl paper an easy sensitive and versatile assay J Immunol Methods 59 269 275 1983 Lye DJ and Birge EA The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates Effect of competing DNA Curr Microbiol 6 139 143 1981 Maniatis T et al Molecular Cloning A Laboratory Manual 1st edn Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1982 Nakamura K et al Microassay for proteins on nitrocellulose filter using protein dye staining procedure Anal Biochem 148 311 319 1985 Neuhoff V et al Spot analysis for glycoprotein determination in the nanogram range Hoppe Seylers Z Physiol Chem 362 1427 1434 1981 Olmsted JB Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples J Biol Chem 256 11955 11957 1981 Palfree RG and Elliott BE An enzyme linked immunosorbent assay ELISA for detergent solubilized la glycoproteins using
22. e TTBS wash solution to each well Adjust the flow valve to the vacuum position and pull the wash solution through the membrane Disconnect the vacuum as soon as the wash solution has drained from all the sample wells Repeat the wash step If the membrane is to be removed from the apparatus prior to performing an immunoassay remove it at this point Otherwise proceed to step 7 Note For better results with Zeta Probe use 0 3 Tween 20 7 Open the flow valve to air Add 100 pl of primary antibody solution to each sample well Allow gravity filtration to occur until the antibody solution has completely drained from the sample wells approximately 30 40 minutes 8 Apply vacuum to the apparatus to remove any excess liquid from the sample wells Open the flow valve to the atmosphere and add 200 400 ul of TTBS wash solution to each well Apply vacuum until the wash solution is drained from the wells Repeat for a total of three wash cycles 10 With the vacuum off and the flow valve open to air add 100 ul of secondary antibody solution to each well Allow gravity filtration to occur 30 40 minutes until all solution has drained from the wells 11 Turn the vacuum on and drain the wells Add 200 400 ul of TTBS wash solution to each well and drain completely Repeat for a total of two washes Note At this point the membrane is ready for development Color development of enzyme conjugated antibodies can be performed in the apparatus or
23. e with 500 ul Tris EDTA TE or H O as described in Section 3 1 At this point the unit is ready for sample application Samples and wash solutions should be applied with a standard pipet or a Costar Octapette pipet with the vacuum off and the flow valve open Apply the denatured DNA ina 50 500 yl sample volume Multiple loadings may be performed However best binding and most rapid results occur using minimum sample volumes Fill all wells with the same volume to obtain homogeneous filtration The sample may be pulled through by applying a gentle vacuum or by gravity filtration Notes a method for applying gentle vacuum to the apparatus is to adjust the flow valve to setting 3 Use a finger to cover the valve port exposed to air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve After the sample has filtered through add 500 ul 0 4 M NaOH to each well for Zeta Probe membrane or 2x SSC for nitrocellulose Apply the vacuum by setting the 3 way valve to setting 1 until the sample wells are empty Disassemble the Bio Dot apparatus Remove the blotted membrane and rinse it in 2x SSC Allow the membrane to airdry The Zeta Probe membrane is ready for hybridization immediately after air drying If hybridization is not to be undertaken within 2 days then vacuum bake the blotted Zeta Probe membrane at 80 C for 30 minutes Nitrocellulose membrane must be baked under vacuum for 2 hours at 80 C
24. ee table 1 Section 2 Special Handling Features The Bio Dot apparatus withstands autoclave temperatures for sterilization as well as cleaning with alcohols acids and base solutions 2 1 Autoclaving The Tygon tubing and flow valve cannot be autoclaved All other components of the apparatus withstand the autoclave treatment After repeated autoclaving 725 cycl es the silicone rubber gasket may need replacing The autoclave conditions that should be used are a maximum sterilization temperature of 250 F 121 C for 15 minutes followed by a 1 minute fast exhaust Higher temperatures or increased exposure times will significantly reduce the life of the apparatus Do not autoclave the unit with the thumbscrews tightened as this may cause the unit to warp during exposure to the elevated temperatures 2 2 Chemical Stability The apparatus is stable in acid and base solutions as well as alcohol solutions This feature allows rapid cleanup and sterilization of the apparatus and gaskets The unit is not compatible with polar aromatic or chlorinated hydrocarbons esters and ketones These solvents will cause degradation of the plastic See Table 1 for list of chemical stabilities For color development in the apparatus the unit is compatible with both the methanol used in horseradish peroxidase HRP color development systems and the low concentration of dimethyl formamide DMF used to solubilize the alkaline phosphatase AP color developm
25. empty wells at each step instead of sample or wash solutions If an overnight incubation is desired adjust the flow valve so that the vacuum manifold is closed off from both the vacuum and air before applying samples see Figure 3 In this configuration solutions will remain in the sample wells with less than 10 loss of volume during an ovemight incubation Note that the unit must be kept at a constant temperature during extended incubations If the unit cools more than 10 C 20 F a partial vacuum will build inside the unit and drainage will occur Any particulate in samples or solutions will block the membrane and restrict flow of solutions through the membrane For best results filter or centrifuge samples to remove particulate matter Check the wells after sample has been applied to ensure that there are no air bubbles in the wells Air bubbles will prevent the sample from binding to the membrane Air bubbles may be removed by pipetting the liquid in the well up and down Proper positioning of the flow valve relative to the level of the apparatus is important for proper drainage The speed of filtration is determined by the difference in hydrostatic pressure between the fluid in the sample wells and the opening of the flow valve which is exposed to air If the opening of the flow valve is above the level of the sample wells very little drainage will occur When the flow valve is positioned at a level below the sample wells proper drain
26. ent reagents However high concentrations of DMF will attack the plastic Also the unit is completely compatible with the low concentrations of diethyl pyrocarbonate DEP used as an alternative to autoclaving for elimination of RNase activity Table 1 Chemical Compatibility Chemicals compatible with Bio Dot apparatus Hydrochloric acid Methanol Sulfuric acid Ethanol Phosphoric acid Butanol Glacial acetic acid Isopropyl alcohol Sodium hydroxide Formaldehyde Potassium hydroxide Hydrogen peroxide Ammonium hydroxide Ethylene glycol Heptane Nitric acid 5 acetone in H O Chemicals incompatible with Bio Dot apparatus use voids warranty Ethyl acetate Butyl acetate Acetone Chloroform Trichloroacetic acid Toluene Benzene Methyl ethyl ketone Methylene chloride Section 3 Bio Dot Assembly 3 1 Assembly 1 Clean and dry the Bio Dot apparatus and gasket prior to assembly 2 Place the gasket support plate into position in the vacuum manifold There is only one way to slide the plate into the manifold 3 Place the sealing gasket on top of the gasket support plate The guide pins on the vacuum manifold help align the 96 holes in the gasket over the 96 holes in the support plate Visually inspect the gasket to make sure the holes are properly aligned If the gasket is not centered pull lightly at the corners until it is aligned Sample template with attached sealing screws Sealing gasket O A am lan CE
27. for complete optimal protein binding 2 DNA RNA a Probe is not pure b Blocker shares common sequences with the probe Assess different blockers Use more stringent washes 23 Section 11 Applications and References 12 1 Common Applications Protein Radioimmunoassay RIA enzyme linked immunoassay EIA fluoroimmunoassay FIA of soluble or particulate cellular antigens Cleveland et al 1981 Shen et al 1980 Analysis of enzymes Faulstich et al 1974 Huet et al 1982 Hormone receptor assays Gershoni and Palade 1983 Schafer et al 1974 Immunoglobulin detection Herbrink et al 1982 Wang et al 1980 Hybridoma screening Bennett and Yeoman 1983 Hawkes et al 1982 Horejsi and Hilgert 1983 Kane et al 1982 Locker and Motta 1983 Shen et al 1980 DNA binding proteins Achberger and Whiteley 1981 Allen and Parsons 1979 Karagyozov and Hadjiolov 1982 Lin and Riggs 1975 Lye and Birge 1981 Glycoprotein lectin assays Gershoni and Palade 1983 Neuhoff et al 1981 Viral antigen analysis clinical applications Cleveland et al 1981 Richman et al 1981 1982 Column or gel column fraction monitoring Cunningham 1983 Palfree and Elliott 1982 Shen et al 1980 Antibody purification Olmsted 1981 Total protein microassay Nakamura et al 1985 Nucleic Acid Dot hybridization of DNA RNA for sequence homology sequence abundance etc Kaftos et al 1979 Thomas 1980 Antibody detection of DNA Tron et al 1
28. for details on how to make the appropriate solution 18 Section 9 Solutions for Nucleic Acid Applications 20x SSC 3 M NaCI 0 3 M trisodium citrate FW 294 1 Dissolve 175 0 g NaCl and 88 2 g trisodium citrate in ddH O Adjust volume to 1 L with ddH O 20x SSPE 3 6 M NaCl 0 2 MNa HPO 7H O 0 02 M EDTA Dissolve 210 0 g NaCl 53 6 g NaHPO 7H O 7 44 g EDTA ddH O Adjust volume to 1 L with ddH O TE 10 mM Tris HCl pH 8 0 1 mM EDTA pH 8 0 Dilute 10 ml 1 M Tris HCl pH 8 0 and 4 ml 0 250 M EDTA pH 8 0 to 1 L with ddH O 100x Denhardt s Solution 2 bovine serum albumin 296 polyvinylpyrrolidone 2 Ficoll Dissolve 2 0 g BSA 2 0 g polyvinylpyrrolidone 2 0 g Ficoll in ddH O Adjust volume to 100 ml with ddH O 20 SDS Dissolve 200 0 g SDS in ddH O Adjust volume to 100 ml with ddH O It may be necessary to heat to 65 C to get into solution 1 M NaHPO pH 7 2 Dissolve 134 0 g Na2 HPO 7H O F W 268 07 in ddH O Add 4 ml 85 H PO Adjust volume to 1 L with ddH O 50 Dextran Sulfate 50 dextran sulfate 0 2 sodium azide Dissolve 50 0 g dextran sulfate and 0 2 g sodium azide in ddH O Adjust volume to 100 ml with ddH O Store at 4 C 19 50 Formamide Dilute 50 0 g formamide to 100 ml with ddH O Store at 4 C Immediately before use deionize the required volume by stirring gently for 1 hour with 1 g mixed bed ion exchange resin AG 501 X8 D resin catalog number 142 6425 per 10 ml of formam
29. hed with 200 yl of distilled water to stop the reaction Following this wash step remove the membrane from the apparatus Rinse the membrane in distilled water and allow it to airdry on filter paper 4 2 Special Protein Blot Applications 1 Soluble enzyme substrate reactions and quantitations Perform an immunassay as described in Section 4 1 Prior to color development disconnect the vacuum and close the flow valve Add an equal volume of substrate solution to all wells Visualize positive reactions and record For quantitation withdraw equal aliquots of the soluble substrate reactant from each well and transfer to a plastic disposable microplate Quantitate using Bio Rad s Model 620 video densitometer 2 Assay for particular antigen or target cell antigen a Place a prewetted filter paper Whatman GF B in the Bio Dot apparatus Attach the sample template and tighten the screws Fill all the wells with buffer and apply a vacuum With the vacuum on and the buffer draining retighten the screws The presence of buffer while applying vacuum will help prevent the filter paper from breaking When the buffer is gone turn off the vacuum and close the flow valve b Add 50 ul fetal bovine serum FBS 10 v v in blocking buffer Allow the FBS buffer to incubate for 10 minutes then open the flow valve and filter through by gravity c Add approximately 12 500 target cells in 50 ul FBS buffer to each well Gently pull the solution through the mem
30. ide Filter through coarse filter paper For DNA or RNA Bound to Zeta Probe Membrane Bio Rad Laboratories 1987 1 mM EDTA 796 SDS 0 5 M NaHPO pH 7 2 For DNA or RNA Bound to Zeta Probe Membrane A Wash two times for 30 60 minutes at 65 C in 1 mM EDTA 40 mM NaHPO pH 7 2 5 SDS B Wash two times for 30 60 minutes at 65 C in 1 mM EDTA 0 1x SSC 0 196 SDS 40 mM NaHPO pH 7 2 1 SDS DNA Bound to Zeta Probe Membrane 50 formamide 1 5x SSPE 1 SDS 0 5 nonfat dry milk For DNA Bound to Nitrocellulose Maniatis et al 1982 6x SSC 0 5 SDS 5x Denhardt s solution 100 ug ml denatured salmon sperm DNA 1mM EDTA For DNA Bound to Nitrocellulose A Rinse in 2x SSC 0 5 SDS B Wash at room temperature for 5 minutes in DNA Bound to Nitrocellulose 50 formamide 1x Denhardt s solution 0 1 SDS For RNA Bound to Nitrocellulose Thomas 1980 50 formamide 5x SSC 1x Denhardt s solution 50 mM NaHPO pH 6 5 250 ug ml denatured salmon sperm DNA For RNA Bound to Nitrocellulose A Wash 4 times at room temperature for 5 minutes in 2x SSC 0 1 SDS B Wash two times at 50 C in 2x SSC 0 5 SDS 1mM EDTA 100 ug ml denatured salmon sperm DNA 20 Section 10 Troubleshooting Guide Filtration Apparatus 1 Leakage or Cross Well Contamination a Improper assembly The screws must be retightened under vacuum following the initial assembly Membrane is
31. in a separate reservoir If performing autoradiography remove the membrane dry it on a filter paper wrap it with plastic wrap and expose it to X ray film The best method to remove the membrane from the Bio Dot apparatus is to leave the vacuum on following the last wash step While the vacuum is on loosen the screws and remove the sample template Turn off the vacuum and remove the membrane 12 For color development in a separate vessel remove the membrane and place it in the color development vessel Wash the membrane twice with TBS to remove excess Tween 20 Prepare the color development solution and incubate the membrane in the solution Gently agitate the solution until development is complete When the reaction has developed remove the membrane and rinse it in distilled water to stop the reaction Place the membrane on filter paper to airdry 13 When using HRP color development substrate wash each well twice with 200 ul TBS to eliminate excess Tween 20 This wash step is not necessary when using AP systems or NBT BCIP color development Add 100 200 ul of the color development solution to each well The reagent can be allowed to react while the solution slowly drains by gravity filtration or the reaction time can be extended by closing the flow valve prior to adding the substrate In either application when the color development is completed the excess substrate should be removed by vacuum and all the sample wells should be vacuum was
32. inage during filtration applications This passive filtration is necessary for quantitative antigen binding Each well should be filled with the same volume of sample solution to ensure homogenous filtration of all sample wells Generally it takes 30 40 minutes for 100 ul of the antigen solution to filter through the membrane If antigen is very dilute and it is necessary to ensure that all proteins in the applied sample are filtered through the membrane an optional wash step can be performed To perform this wash add an aliquot of TBS equal to the original sample volume to each sample well Allow this material to passively filter through the membrane by gravity filtration If the membrane is going to be removed from the apparatus following binding of antigen proceed to step 6 and follow the instructions for the wash step The wash step should be performed prior to disassembling the apparatus to ensure that all antigen is removed from the drain ports underneath the membrane After the antigen samples have completely drained from the apparatus add 200 300 ul of the blocking solution to each well Allow gravity filtration to occur until the blocking solution has completely drained from every well This step should take approximately 60 minutes Do not apply vacuum to speed up this step as it will lead to poor assay results 6 Adjust the flow valve so that the vacuum chamber is exposed to air Add 200 400 ul of the Tween tris buffered salin
33. ll slot format sample template The Bio Dot apparatus is simple to operate As shown in figure 1 a sheet of membrane is clamped between the gasket and the 96 well sample template The gasket is aligned above the support plate which is placed over the vacuum reservoir This assembly is attached to a vacuum source by the in line 3 way flow valve which allows on off control of vacuum during assay procedures The entire assembly is held together by the four screws on the sample template and the patented rubber sealing gasket seals prevent well to well leakage whether the vacuum is on or off Sample can be easily applied to the 96 well formate with a standard pipet or with the Costar Octapette pipet The material used in the construction of the Bio Dot blotting apparatus can withstand rigorous sterilization and cleanup procedures The Bio Dot apparatus can be repeatedly autoclaved and is resistant to many chemicals including acids bases and ethanol 1 1 Specifications Materials Bio Dot apparatus Molded polysulfone Bio Dot gasket Silicone rubber Stopcock Polytetrafluoroethylene PTFE Tubing Tygon Shipping weight 600 grams Overall size 13x 15x 6 cm Membrane size 12 x 9 cm sheet Autoclaving 15 minutes at 250 F 121 C with a 1 minute fast exhaust Chemical compatibility The Bio Dot apparatus can be used with 100 alcohol solutions and concentrated alkali or acid solutions It cannot be used with aromatic or chlorinated hydrocarbons S
34. much sample is present wicking into the membrane around the well will occur Use serial dilutions of the samples to determine optimal amounts to load Il Poor Binding to Membrane 1 Nitrocellulose a DNA RNA vwill only bind efficiently in 20x SSC or 1 M ammonium acetate Use the Zeta Probe membrane as an alternative DNA must be single stranded and RNA must be denatured DNA 500 bp may not bind to nitrocellulose Use the Zeta Probe membrane as an alternative Mixed ester cellulose binds DNA RNA and protein very poorly Use Bio Rad s pure nitrocellulose 21 d Proteins 15 000 daltons may show decreased binding to 0 45 um nitrocellulose Use the Zeta Probe membrane or 0 2 um nitrocellulose Also glutaraldehyde fixation will increase retention of small proteins and peptides to both nitrocellulose and the Zeta Probe membrane Protein may be removed from nitrocellulose by SDS NP 40 or Triton X 100 Use Tween 20 in washes Reduce concentrations or time of any SDS or NP 40 washes Ill High Background After Incubation With Labeled Probes 1 DNA and RNA a Unincorporated label small radioactive decay products and small probe fragments resulting from nicktranslation can increase overall background Use the Bio Spin chromatography columns to remove unincorporated label Filter hybridization solutions before use Use the probe as soon as possible after preparation Reduce exposure of the probe to DNase during nicktranslatio
35. n Improper blocking conditions were used Increase the blocker concentration Use a different heterologous nucleic acid in the prehybridization mixtures Sonicate the solution thoroughly and denature before use The blocker shares common sequences with host or vector of cloned probe Vary the blocker Yeast tRNA may be useful instead of salmon sperm DNA Cut the probe out of vector and purify Washes were insufficient Include stringent washes i e increase the temperature of the washes or decrease the salt concentration Increase the number and the length of the standard washes The probe was too hot or concentrated Dilute the probe The incubation period was too long Shorten the reaction time The bag used in hybridization collapsed on the membrane Be sure the membrane is floating freely in the hybridization bag and that the volume of solution present is enough to prevent the bag from collapsing during incubations Dust was present on the membrane Remove by washing in 2x Denhardt s prior to baking or with a brief wash prior to hybridization The gasket is contaminated by radioactivity Replace the gasket 2 Protein a Impure secondary antibody was used Use Bio Rad s affinity purified blotting grade second antibody Excessive reaction time in the substrate Remove the blot from the substrate reaction when the signal to noise level is acceptable Improper blocking conditions were used Be sure the blocker is pure protei
36. n Increase the blocker concentration or blocking time Match the blocker with the detection system Hemoglobin reacts with horseradish peroxidase BSA may contain IgG contaminants Primary or secondary antibody is too concentrated Dilute the antibodies Washes were insufficient Increase the number and or duration of the washes Include progressively stronger detergents in the washes For example SDS2NP 40 Tween 20 Also include Tween 20 in the antibody buffers to reduce nonspecific binding 22 IV Poor Detection Sensitivity or No Reactivity 1 DNA RNA a This problem may occur when total genomic DNA is probed for single copy or low copy number genes Try the Zeta Probe membrane for binding and retention of increased quantities of DNA b Hybridization was insufficient Incorporate 1096 dextran sulfate in the hybridization mixture This polymer effectively reduces the solvent volume thereby increasing the concentration of the solutes and enhancing hybridization Exposure time was insufficient Increase the time of exposure Sample load was insufficient Increase the sample load e Probe concentration is too low If low signal is accompanied by low background then the probe concentration can be increased f Binding of nucleic acid to the membrane was incomplete See Troubleshooting Part Il g If no autoradiographic signal is seen make sure the probe was denatured by heating to 100 C exposure to 0 4 N NaOH or by heating
37. ne prior to placing it in the apparatus Nitrocellulose membranes are prewetted in TBS nylon membranes such as the Zeta Probe membrane are prewetted in distilled water see Section 9 for solution preparation Make sure that all the screws have been tightened under vacuum to ensure that there will not be any cross well contamination Notes Zeta Probe membranes must be removed from the Bio Dot apparatus after the antigen is immobilized The blocking and other incubation steps should be carried out in a separate container Zeta Probe membranes require more stringent blocking conditions using 5 w v BLOTTO or 3 w v gelatin in 1x TBS which cannot be filtered through the membrane using the Bio Dot apparatus Rehydrate the membrane to ensure uniform binding of the antigen Use 100 ul TBS per well for nitrocellulose membranes Use 100 pl distilled water per well for Zeta Probe membranes Adjust the flow valve so that the vacuum chamber is open to air flow valve setting 2 Figure 3 Fill the appropriate wells with antigen protein solution using any volume up to 500 ul per well Multiple applications of antigen to a sample well are possible but the most rapid and efficient use of the apparatus is achieved by applying the required amount of antigen in a minimal sample volume Allow the entire sample to filter through the membrane by gravity flow Make sure that the flow valve is positioned at a level below the sample wells to ensure proper dra
38. nitrocellulose membrane discs J Immunol Methods 52 395 408 1982 Ricciardi RP et al Purification and mapping of specific mRNAs by hybridization selection and cell free translation Proc Natl Acad Sci USA 76 4927 4931 1979 Richman DD et al A rapid radioimmunoassay using I labeled staphylococcal protein A for antibody to varicella zoster virus J Infect Dis 143 693 699 1981 Richman DD et al A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus J Med Virol 9 299 305 1982 Schafer A et al A solid phase radioimmunoassay for urine aldosterone using antibodies linked to nylon nets FEBS Lett 48 230 234 1974 Shen V et al Monoclonal antibodies to Escherichia coli 50S ribosomes Nucleic Acids Res 8 4639 4649 1980 Smith LH et al Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas Am J Obstet Gynecol 166 634 645 1992 Thomas P Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose Proc Natl Acad Sci USA 77 5201 5205 1980 Tron F et al Clinical and theoretical interest of the immunochemical analysis of antinuclear antibodies in systemic lupus erythematosus Adv Nephrol Necker Hosp 12 215 237 1983 Wang R et al A simplified solid phase immunofluorescence assay for measurement of serum immunoglobulins Clin Chim Acta 102 169 177 1980 Winberg G and Hammarskjold ML I
39. not properly rehydrated after assembly Always rehydrate the membrane prior to applying samples Apply vacuum only until solutions are removed from the sample wells then disconnect the vacuum source 2 No Filtration or Uneven Filtration Occurring a Macromolecular polymers cellular debris or dirt is plugging the membrane Centrifuge samples prior to application to remove particulates Filter solution prior to use to ensure removal of particulate material Cover wells with Parafilm during lengthy incubations Bubbles are obstructing the filtration Use a needle to break any bubbles being careful not to puncture the membrane Pipet liquid in the wells up and down to displace bubbles The flow valve is positioned higher than the apparatus The flow valve must be lower than the level of the sample wells on the apparatus for proper drainage to occur Improper blocking reagent is used BSA is the recommended blocker for nitrocellulose gelatin will plug the apparatus and no filtration will occur The Zeta Probe membrane which requires more stringent blocking with BLOTTO or with gelatin and MPO should be removed from the Bio Dot apparatus following antigen immobilization and the rest of the assay should be conducted in a separate container 3 Halos a b Membrane is not properly rehydrated before applying samples Always rehydrate membrane prior to applying any sample Excessive concentrations of sample are loaded When too
40. ose In that case add 1096 dextran sulfate to the hybridization solution Note formamide can also be used in the hybridization buffer to lower the incubation temperature when binding DNA to nitrocellulose or Zeta Probe membrane Maniatis et al 1982 Casey and Davidson 1977 For alternative protocols see the Zeta Probe Membrane Instruction Manual Add the denatured probe remove all air bubbles and reseal the bag Mix the contents of the bag Hybridize with agitation For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 4 24 hours at 65 C 4 24 hours at 68 C 4 24 hours at 42 C Carefully remove the hybridization solution by cutting one corner Remove hybridized Zeta Probe membrane from the plastic bag Note Once hybridizationhas begun do not let the membrane dry Washes 1 Agitate the solutions when washing membranes For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose A Wash two times for 30 60 A Rinse in A Wash 4 times at room minutes at 65 C in 2x SSC 0 5 SDS temperature for 5 minutes 1 mM EDTA in 2x SSC 0 1 SDS 40 mM NaHPO pH 7 2 5 SDS B Wash two times for 30 60 B Wash at room B Wash two times at minutes at 65 C in temperature for 50 C in 1 mM EDTA 5 minutes in 2x SSC 0 5 SDS 0 1x SSC 0 1 SDS 1mM EDTA 40 mM NaHPO pH 7 2 1 SDS After washing the blotted membrane
41. solation of DNA from agarose gels using DEAE paper Application to restriction site mapping of adenovirus type 16 DNA Nucleic Acids Res 8 253 264 1980 Ficoll is a trademark of Amersham Biosciences Parafilm is a trademark of American National Can Co Triton is a trademark of Union Carbide Chemicals amp Plastics Technology Corp Tween is a trademark of ICI Americas Inc 26 Section 12 Ordering Information Catalog Description 170 6545 Bio Dot Microfiltration Apparatus includes Bio Dot sample template vacuum manifold base plate membrane support and gasket 170 6547 Bio Dot Module includes Bio Dot sample template membrane support and gasket 170 6546 Bio Dot Gaskets 3 gaskets per package 170 6542 Bio Dot SF Microfiltration Apparatus includes Bio Dot SF sample template vacuum manifold base plate membrane support gasket and filter paper 170 6543 Bio Dot SF Module includes Bio Dot SF sample template membrane support gasket one conversion of the Bio Dot apparatus to the Bio Dot SF apparatus 170 6544 Bio Dot SF Gaskets 2 gaskets per package 162 0161 Bio Dot SF Filter Paper 60 sheets 162 0117 Nitrocellulose Membrane 0 45 um for Bio Dot SF applications 9 x 12 cm sheets 10 162 0153 Zeta Probe Membrane for use with the Bio Dot apparatus and Bio Dot SF apparatus 9 x 12 cm sheets 15 732 6000 Bio Spin 6 columns 732 6004 Bio Spin 30 columns 142 6425 AG 501 X8 D resin H OH 20 50 mesh 500 g 27
42. to 65 C for 5 minutes in 5096 formamide prior to hybridization 2 Protein a Antigen binding was incomplete See Troubleshooting Part Il b Monoclonal antibodies may not recognize a denatured antigen Asses the binding of other monoclonal or polyclonal antibodies Blot only native proteins c The enzyme conjugate or the substrate is inactivated Primary or secondary antibody is inactive or nonsaturating Test the enzyme antibody and substrate separately for activity Increase concentration of the primary or secondary antibody Eliminate the detergents from reactions and washes With HRP avoid sodium azide as it is a potent inhibitor of the enzyme d Forlabeled probes exposure time was insufficient Increase the time of exposure e Antibody reaction times are insufficient Increase reaction times f Sample load was insufficient Increase the concentration of antigen applied V Nonspecific or Nonquantitative Detection 1 Protein a Monoclonal antibodies may react nonspecifically with SDS denatured proteins Compare binding of other monoclonals or polyclonal antibodies Blot native proteins b Concentration of the primary or secondary antibody is excessive Increase the dilution of the antibodies c Primary or secondary antibody is contaminated with nonspecific or speciescross reactive IgG Use a purified IgG primary antibody fraction and affinity purified blotting grade secondary antibody d Slow gentle filtration is needed
43. urs at 42 C Hybridization 1 Immediately before use fragment and denature the probe and carrier DNA as follows Add to the precipitated RNA probe 0 1 ml of yeast RNA 20 mg ml 0 5 ml of carrier DNA 10 mg ml and 0 6 ml of deionized formamide mix thoroughly and heat at 70 C for 5 minutes 2 Cut one corner of the bag remove the prehybridization solution and replace it with hybridization solution DNA Bound to DNA Bound to Zeta Probe Membrane Nitrocellulose 5096 formamide 5096 formamide 1 5x SSPE 1x Denhardt s solution 1 SDS 0 1 SDS 0 5 nonfat dry milk 100 ug ml denatured salmon sperm DNA 3 Add probe then seal the open corner taking care to exclude all air bubbles Mix the contents of the bag thoroughly Incubate at 50 C for 4 24 hours Note After beginning hybridization the membranes should not be permitted to dry 15 Washes 1 At the completion of hybridization remove the membranes from their hybridization bags into 2x SSC Rinse briefly then wash them sequentially with agitation for 15 minutes at room temperature in the following solutions a 2x SSC 0 1 SDS b 0 5x SSC 0 1 SDS C 0 1x SSC 0 1 SDS For DNA bound to nitrocellulose membranes it may be necessary to include an RNase treatment in the wash Membranes are treated with 20 ug ml RNase for 30 minutes at 37 C in 2x SSC Santzen et al 1986 After washing the blotted membranes are ready for autoradiography If no further cycles of hybridization
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