Bio-Dot® SF Microfiltration Apparatus Instruction Manual
Contents
1. Bio Dot SF Microfiltration Apparatus Instruction Manual Catalog Number 170 6542 170 6543 For technical service call your local Bio Rad office or in the U S call 1 800 4BIORAD 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 Section 12 Section 13 Table of Contents Page Introduction eth eter eto e Por reped 1 SPSCHICATIONS nep 1 Special Handling and Features sssssssssssss 1 AULOCIAVING atit peret E eei ee et e C de Es 1 Chemical Stability sess 1 Blo Dot SF Assembly ctore eot prts els 3 ASSOImbly ssiceetusi re tetdteu teu d rer a tue a uf coles pee des 3 Helptul Hints 2 cite Den Let c eder 5 Protein Slot Blotting sssssee 6 General Recommendations esses 6 Immunoassay Proce dUi Gesce eera e R RE 6 DNA Slot Blotting iere ead caede nerd 7 RNA Slot Blotting oett ote Rate in 8 Alkaline RNA Denaturation and Fixation ssessssssesssss 8 Glyoxal RNA Denaturation and Fixation sssssssesess 9 Hybridization Protocols for Nucleic Acids 10 Probe Recommendations ssssssssssseseeeeeneereenn ene 10 Hybridization Protocols for DNA or RNA Bo2. 24 hours Note Once hybridization has begun do not allow the membrane to dry 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 e 2x SSC 0 1 SDS 0 5x SSC 0 1 SDS e 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 Johnson et al 1984 12 3 After washing the blotted membranes are ready for autoradiography If no further cycles of hybridization 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 wet membranes will stick to the film and moisture on the film will cause black spots Note To increase the rate of hybridization include 10 dextran sulfate final concentration in the hybridization solution Maniatis et al 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 P
3. An enzyme linked immunosorbent assay ELISA for detergent solubilized la glycoproteins using 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 125 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
4. Improper blocking conditions were used Be sure the blocker is pure protein Increase the blocker concentration or blocking time Match the blocker with the detection system i e hemoglobin reacts with horseradish peroxidase bovine serum albumin 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 SDS NP 40 Tween 20 Also include Tween 20 in the antibody buffers to reduce nonspecific binding Poor Detection Sensitivity or No Reactivity 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 d 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 i
5. 2 Glyoxal RNA Denaturation and Fixation 1 Prepare RNA samples to the following final concentrations 50 dimethyl sulfoxide DMSO 10 mM NaH PO pH 7 0 1 M glyoxal 2 Incubate the RNA for 1 hour at 50 C Cool the samples on ice 3 Pre wet the blotting membrane by placing it gently at a 45 angle into a tray of wetting solution Zeta Probe membrane is wetted in distilled water nitrocellulose is wetted in 6x SSC see Section 9 for solution preparation Always wear gloves when handling blotting membranes 4 Assemble the Bio Dot SF apparatus according to the instructions in Section 4 1 Remember to apply the vacuum and then retighten the screws that hold the apparatus together 5 Samples and wash solutions may be applied with a standard pipet or an 8 channel 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 6 Rinse all wells to wash any sample on the side of the wells through Rinse with 500 ul TE Apply vacuum flow valve setting 1 Figure 3 until the sample wells are dry Disassemble the Bio Dot SF apparatus Remove the blotted membrane Remove glyoxal adducts by pouring 20 mM Tris HCl pH 8 0 1 mM EDTA heated to
6. 7 0 to the target DNA solution Leave DNA on ice while preparing Bio Dot Prewet 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 see Section 9 for recipes Zeta Probe membranes should be wetted in distilled water Assemble the Bio Dot SF apparatus according to the instructions in Section 3 1 Apply the vacuum and then retighten the screws that hold the apparatus together Rehydrate the membrane with 500 ul Tris EDTA TE or H2O 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 an 8 channel pipet with the vacuum off and the flow valve open Apply the denatured DNA in a 50 500 ul sample volume The recommended sample loading volume is at least 200 pl If sample volumes of less than 200 ul are applied they must be very carefully applied to the center of the well Applying the solution on one side of the well results in unequal distribution of DNA This may result in unevenly shaped bands leading to distorted densitometer readings Fill all wells with the same volume to ensure homogeneous filration The sample may be pulled through by applying a gentle vacuum or by gravity filtration Note A method for applying gentle vacuum to the apparatus is to adjust the flow valve to valve setting 3
7. 95 C onto the membrane and agitating at room temperature until the solution cools Alternatively place the membrane in a vacuum oven at 80 C for 1 hour for Zeta Probe membrane 2 hours for nitrocellulose and omit step 8 9 Nitrocellulose membranes must be baked under vacuum for 2 hours at 80 C before hybridization If hybridization is not to be undertaken within 2 days 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 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 ratio during hybridization For hybridization on Zeta Probe membrane the following is recommended Probe specific activity 108 cpm ug probe Probe concentration in the hybridization mixture 106 counts ml 10 50 ng 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 cl
8. and 100 ml is used for conjugate dilution Primary antibody solution 100 ml Same as nitrocellulose membrane solution Secondary antibody solution 100 ml Same as nitrocellulose membrane solution Color development solution Same as nitrocellulose membrane solution 14 Section 9 Solutions for Nucleic Acid Applications For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 1 mM EDTA 6x SSC 5096 formamide 796 SDS 0 596 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 1 mM EDTA salmon sperm DNA 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 ddH2O Adjust volume to 1 L with ddH2O 20x SSPE 3 6 M NaCl 0 2 M NagHPO 7H50 FW 268 07 0 02 M EDTA Dissolve 210 0 g NaCl 53 6 g NagHPO4 7H20 7 44 g EDTA in ddH20 Adjust volume to 1 L with ddHoO 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 ddH2O 100x Denhardt s Solution 296 bovine serum albumin BSA 296 polyvinylpyrrolidone 296 Ficoll Dissolve 2 0 g BSA 2 0 g polyvinylpyrrolidone 2 0 g Ficoll in ddH2O Adjust volume to 100 ml with ddH5O 2096 SDS Dissolve 20 0 g SDS in ddH5O Adjust volume to 100 ml with ddH90 It may be necessary to heat to 65 C to ge
9. fraction and affinity purified blotting grade secondary antibody d Slow gentle filtration is needed 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 Section 11 Legal Notices Tween is a trademark of ICI Americas Inc Ficoll is a trademark of Pharmacia Biotech AB Teflon is a trademark of E DuPont de Nemours amp Co Triton is a trademark of Union Carbide Chemicals amp Plastics Technology Corp Tygon is a trademark of Norton Co Parafilm is a trademark of American National Can Co Section 12 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 Bio Rad Laboratories Immun Blot Instruction Manual Bio Rad Laboratories Zeta Probe Membrane Instruction Manual Brandsma J and Miller G Nucleic acid s
10. of serum immunoglobulins Clin Chim Acta 102 169 177 1980 Winberg G and Hammarskjold ML Isolation 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 Section 13 Ordering Information Catalog Description 170 6542 Bio Dot SF Apparatus 170 6543 Bio Dot SF Module for Bio Dot to Bio Dot SF conversion 170 6544 Bio Dot SF Gaskets 2 162 0161 Bio Dot Bio Dot SF Filter Paper 11 3 x 7 7 cm 60 sheets 170 6545 Bio Dot Apparatus 170 6546 Bio Dot Gaskets 3 170 6547 Bio Dot Module for Bio Dot SF to Bio Dot conversion 162 0117 Nitrocellulose Membranes 0 45 um 9 x 12 cm 10 162 0153 Zeta Probe Membranes 9 x 12 cm 15 162 0190 Zeta Probe GT Membranes 9 x 12 cm 15 732 6002 Bio Spin b 25 pk 142 6425 AG 501 X8 D Resin Ht OH 20 50 mesh 500 g 21 Iz epo p Bio Rad Laboratories Inc Life Science Web site www bio rad com USA 800 4BIORAD Australia 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 2527 3454 Canada 905 712 2771 China 86 21 63052255 Czech Republic 420 2 41 43 05 32 Denmark 44 52 10 00 Finland 09 804 22 00 Gr oup France 01 47 95 69 65 Germany 089 318 84 0 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 6398112 113 114 6450092 93 Israel 03 951 4127 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Latin America 305 894 5950 Mexico 55 52 00 05 20 The Netherlands 0318 540666
11. 8 rows and 6 columns Sample can be applied using a standard pipet or with an 8 channel pipet The material used in the construction of the Bio Dot SF blotting apparatus can withstand rigorous sterilization and cleanup procedures The Bio Dot SF apparatus can be repeatedly autoclaved and is resistant to many chemicals including acids bases and ethanol 1 1 Specifications Materials Bio Dot SF apparatus Molded polysulfone Bio Dot SF gasket Silicone rubber Stopcock Teflon Shipping weight 600 grams Overall size 13x15x6cm 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 SF apparatus can be used with 100 alcohol solutions and concentrated alkali or acid solutions It cannot be used with aromatic or chlorinated hydrocarbons See Table 1 Section 2 Special Handling and 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 cycles 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 significant
12. New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 095 721 1404 Singapore 65 6415 3188 South Africa 00 27 11 4428508 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 9555 Taiwan 8862 2578 7189 2578 7241 United Kingdom 020 8328 2000 Sig 1103 M1706542 Rev C
13. 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 SF apparatus Remove the blotted membrane and rinse it in 2x SSC Allow the membrane to air dry 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 membranes must be baked under vacuum for 2 hours at 80 C 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 Section 6 RNA Slot 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 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 9 for solution prepar
14. after preparation Reduce exposure of the probe to DNase during nick translation 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 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 17 2 Protein a Impure secondary antibody was used Use 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
15. ation Assemble the Bio Dot SF 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 4 Samples and wash solutions may be applied with a standard pipet or an 8 channel 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 three 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 5 Rinse all wells to wash any sample on the side of the wells through 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 6 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 C 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
16. bbles 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 where it is at a level below the sample wells proper drainage will occur during filtration applications The recommended sample loading volume is at least 200 pl If sample volumes of less than 200 ul are loaded they must be carefully applied to the center of the well Applying the solution on one side of the well results in unequal distribution of sample This results in unevenly shaped bands leading to distorted densitometer readings 7 The Bio Dot SF apparatus is designed for use with an 8 channel pipet allowing eight sample or wash solutions to be quickly and easily applied to one row at a time 8 The best method for removing the blotted membrane from the Bio Dot SF apparatus is to leave the vacuum on following the wash step With the vacuum applied loosen the screws and remove the sample template Next turn off the vacuum and remove the membrane Section 4 Pro
17. 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 8 2 Solutions for Zeta Probe Membrane Two methods of blocking are given Method A uses nonfat dry milk as the blocking agent Method B uses gelatin and MPO as the blocking agents The solutions for the two methods are not interchangeable If Method A is chosen all solutions must be prepared 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 2 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 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 which 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
18. d 10 for solution preparation A 10 minute soak is recommended for complete wetting of the membrane to ensure 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 filter paper in the apparatus so it extends over the edges of the filter paper For the best slot blot results use membrane sheets that have been precut to a 9 x 12 cm size catalog number 162 01 17 for nitrocellulose 162 0153 for Zeta Probe membrane In all cases the membrane should not extend beyond the edge of the gasket after the Bio Dot SF apparatus is assembled Remove any air bubbles trapped between the membrane and the filter paper Note PVDF membrane is not recommended 5 Fig 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 1 3 o Outlet port 4 2 2 Diagonal crossing pattern for tightening screws in the Bio Dot apparatus Attach a vacuum source house vacuum or vacuum pump to the flow valve with a waste trap set up and positioned between the vacuum outlet and flow valve Turn on the vacuum and set the 3 way valve to apply vacuum t
19. e assay for the detection of antibodies J Immunol Methods 48 293 298 1982 Holland LJ 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 I 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 bind
20. eanup method is by column chromatography This can be done quickly and easily with the Bio Spin chromatography columns Bio Spin 6 columns 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 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 your application For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 1 mM EDTA 6x SSC 5096 formamide 796 SDS 0 596 SDS 5x SSC 0 5 M NaHPO pH 7 2 5x Denha
21. ide 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 50 formamide 50 formamide 1 5x sodium sodium phosphate EDTA SSPE 0 1 SDS 1 SDS 5x SSPE 0 5 nonfat 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 carrier 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 Seal the bag and incubate DNA Bound to Zeta Probe Membrane DNA Bound to Nitrocellulose 30 minutes at 50 C 4 hours 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 Cut one corner of the bag remove the prehybridization solution and replace it with hybridization solution DNA Bound to Zeta Probe Membrane DNA Bound to nitrocellulose 50 formamide 50 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 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
22. ilter Apparatus 1 Leakage or cross well contamination a Improper assembly The screws must be retightened under vacuum following the initial assembly The thickness of filter paper must be correct or leakage will result Exactly three sheets of filter paper must be placed on the membrane support Do not use filter paper other than the Bio Dot SF filter paper Membrane is 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 b Bubbles are obstructing the filtration Pipet liquid in the wells up and down to displace bubbles Use a needle to break any bubbles being careful not to puncture the membrane c 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 3 Halos a Membrane is not properly rehydrated before applying samples Always rehydrate membrane prior to applying any sample 16 b Use of more than three filter paper sheets or more than one membrane wil
23. ing J Biochem Biophys Methods 5 329 339 1982 Kranz RG and Gennis RB A quantitative radioimmunological 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 Locker D Motta G Detection of antibody secreting hybridomas with diazobenzyloxymethy 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 20 Palfree RG and Elliott BE
24. ing 3 Ir The manifold is exposed to both air and the vacuum Use this setting for gentle vacuum applications where the amount of vacuum is regulated by putting a finger over the port Bio Dot yaad exposed to the atmosphere Fig 3 Optional settings for the 3 way flow valve to obtain optimal performance from the Bio Dot SF 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 source If some sample wells are not used in a particular assay those wells must be closed off to insure 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 empty wells at each step instead of sample or wash solutions 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 insure that there are no air bu
25. l cause lateral diffusion and leakage Excessive concentrations of sample are loaded When too 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 ll Poor Binding to Membrane 1 Nitrocellulose a DNA RNA will 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 Zeta Probe membrane as an alternative Mixed ester cellulose binds DNA RNA and protein very poorly Use Bio Rad s pure nitrocellulose Proteins 15 000 daltons may show diminished 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 Zeta Probe membrane Protein may be removed from nitrocellulose by SDS NP 40 Triton X 100 Use Tween 20 in washes Reduce concentrations or time of any SDS or NP 40 washes High Background After Incubation with Labeled Probes DNA and RNA a Unincorporated label small radioactive decay products and small probe fragments resulting from nick translation 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
26. ly 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 both acid and base solutions It is stable in all concentrations of alcohol solutions Both of these features allow 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 stability For color development in the apparatus the unit is compatible with both the methanol used in the horseradish peroxidase HRP color development systems and the low concentration of DMF used to solubilize the alkaline phosphatase AP color development reagents However high concentrations of DMF will attack the plastic Also the unit is completely compatible with the low concentrations of diethyl pyrocarbonate DEPC used as an alternative to autoclaving for elimination of RNase activity Table 1 Chemical Compatibility Chemicals compatible with Bio Dot SF apparatus Hydrochloric acid Methanol Sulfuric acid Ethanol Phosphoric acid Butanol Glacial acetic acid sopropanol Sodium hydroxide Formaldehyde Potassium hydroxide Hydrogen peroxide Ammonium hydroxide Ethylene glycol Heptane 5 acetone in H2O Nitric acid Chemicals that will attack poly
27. ng it in the apparatus Nitrocellulose membranes are prewetted in TBS Zeta Probe membrane is prewetted in distilled water see Section 10 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 Rehydrate the membrane to ensure uniform binding of the antigen Use 100 ul TBS per well for nitrocellulose membranes Use 100 ul distilled water per well for the Zeta Probe membrane 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 applying 50 500 ul per well The recommended sample loading volume is at least 200 pl If less than 200 ul is applied the sample must be carefully applied to the center of the well Applying the solution on one side of the well results in unequal distribution of sample This results in unevenly shaped bands leading to distorted densitometer readings 10 11 12 13 Note The solution applied should be free of insoluble particles to avoid clogging of wells Allow the entire sample to filter through the membrane by gentle vacuum Make sure that the flow valve is positioned at a level below the sample wells to ensure proper drainage during filtration applications Slow gentle filtration is necessary for quantitative antigen binding Each well should be filled with the same volume of sample solution to ensure homogene
28. o the apparatus flow valve setting one 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 flow valve setting two Figure 3 Apply 100 ul to all the sample wells Use of an 8 channel pipet and buffer reservoirs see Section 13 for information will simplify the process of adding solutions to the Bio Dot SF 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 three 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 vacuum At this point the unit is ready for sample application Flow Valve Setting 1 Air The vacuum manifold is exposed to the vacuum source only Use for applying vacuum to the Bio Dot SF apparatus Bio Dot Vacuum Flow Valve Setting 2 Air The manifold is exposed to air Use for gravity filtration procedures Bio Dot Vacuum Flow Valve Sett
29. on periods may increase the sensitivity of detection in some situations Remove unbound primary antibody by washing the membrane in TTBS for 5 minutes with gentle agitation Decant the wash solution and repeat the wash step Transfer the membrane to the secondary antibody conjugate solution Transfer the membrane from the conjugate solution and wash it in TTBS for 5 minutes Decant the buffer and repeat the wash step Prior to color development do a final 5 minute wash in TBS to remove residual Tween 20 detergent from the membrane surface Do the color development reaction according to the Immun Blot assay kit instruction manual If performing autoradiography remove the membrane dry it on a filter paper wrap it with plastic wrap and expose it to X ray film Section 5 DNA Slot Blotting This section gives protocols for DNA slot blotting The alkaline blotting method using Zeta Probe membrane and a more standard method for DNA blotting to nitrocellulose are 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
30. ous filtration of all sample wells Wash each sample well with at least 200 ul of TBS Pull the wash liquid through by applying gentle vacuum flow valve setting 3 Figure 3 After the wells have completely drained remove the membrane from the apparatus The best method for removing the membrane from the Bio Dot SF apparatus is to leave the vacuum on following the wash step With the vacuum applied loosen the screws and remove the sample template Next turn off the vacuum and remove the membrane Place the membrane in the blocking solution The blocking solution used is dependent on the type of membrane used Nitrocellulose is blocked in gelatin BSA or non fat dry milk Zeta Probe membrane is blocked in either nonfat dry milk or gelatin and 1 methyl 2 pryrrolidinone MPO see Section 10 solution preparation All solution volumes should be at least 0 5 ml cm of membrane Larger volumes may be used for convenience Gently agitate the solution using a shaker platform Continue the incubation for 30 minutes to 1 hour Remove the membrane from the blocking solution and transfer to a dish or tray containing Tween Tris buffered saline TTBS Wash for 5 minutes with gentle agitation Decant the wash solution Repeat the wash step Transfer the membrane from the TTBS wash solution to a dish or tray containing the primary antibody solution Incubate 1 to 2 hours with gentle agitation Overnight incubation may be desirable since longer incubati
31. pot hybridization rapid quantitative screening of lymphoid cell lines for Epstein Barr 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 19 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 sensitiv
32. rdt s solution 1x Denhardt s solution 100 ug ml denatured 50 mM NaHPO pH 6 5 salmon sperm DNA 250 ug ml denatured 1 mM EDTA salmon sperm DNA 10 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 nitrocellulose In that case add 10 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 2 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 3 Carefully remove the hybridization solution by cutting one corner Remove hybridized membrane f
33. robe 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 2 times 20 minutes each in a large volume of 0 1x SSC 0 5 SDS at 95 C 2 Check membrane for removal of autoradiaography patterns by overnight exposure Section 8 Solutions for Protein Applications 8 1 Solutions for Nitrocellulose Membrane Tris Buffered Saline 1x TBS 2 L 20 mM Tris pH 7 5 500 mM NaCl Dissolve 4 84 g Tris 58 48 g NaCl in ddH O Adjust to pH 7 5 with HCl Adjust the volume to 2 L with ddH O Tween 20 Wash Solution 1x TTBS 1 L 20 mM Tris pH 7 5 500 mM NaCl 0 0596 Tween 20 Add 0 5 ml Tween 20 to 1 L of TBS Blocking Solution 100 ml 3 gelatin TBS Add 3 0 g gelatin to 100 ml TBS Heat at 37 C to dissolve the gelatin Antibody Buffer 200 ml 1 gelatin TTBS Add 2 g gelatin to 200 ml TTBS Heat at 37 C to dissolve the gelatin 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 Bio Rad s species specific secondary antibody conjugate 1 3 000 by adding 33 pl of conjugate to 100 ml of antibody buffer 13 Color Development Solution The specific chemicals and
34. rom the plastic bag Note Once hybridization has 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 2 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 2 times for 30 60 B Wash at room B Wash 2 times at 50 C in minutes at 65 C in temperature for 5 minutes 0 1x SSC 0 1 SDS 1 mM EDTA in 2x SSC 0 5 SDS 40 mM NaHPO pH 7 2 1 SDS C Wash at 68 C 2 times for 1 hour each in 0 1x SSC 0 5 SDS 2 After washing the blotted membrane 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 or enclosed in a sealable plastic bag Do not allow the wet membrane to come in contact with the film because wet membranes will stick to the film and any moisture on the film will cause black spots 11 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 ins
35. s seen make sure the probe was denatured by heating to 100 C exposure to 0 4 N NaOH or by heating to 65 C for 5 minutes in 50 formamide prior to hybridization Protein a Antigen binding was incomplete See Troubleshooting Part Il b Monoclonal antibodies may not recognize a denatured antigen Assess 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 first or second 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 Sample load was insufficient Increase the concentration of antigen applied 18 V Nonspecific or Nonquantitative Detection Protein a Monoclonal antibodies may react nonspecifically with SDS denatured proteins Compare binding of other monoclonals or polyclonal antibodies Blot native protein 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 species cross reactive IgG Use a purified IgG primary antibody
36. sulfone Ethyl acetate Toluene Butyl acetate Benzene Acetone Methyl ethyl ketone Chloroform Methylene chloride Trichloroacetic acid Section 3 Bio Dot SF Assembly 3 1 Assembly 1 Clean and dry the Bio Dot SF 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 Place the sealing gasket on top of the vacuum manifold P Sample template with attached sealing screws Membrane Filter paper 3 sheets Sealing gasket Gasket support plate Vacuum manifold P 2 Tubing and flow valve M ME Fig 1 Diagram of proper Bio Dot SF apparatus assembly 3 Moisten three sheets of Bio Dot SF filter paper catalog number 162 0161 in wetting solution Use the same solution that is used to prewet the membrane step 4 Place the three sheets onto the membrane support The filter paper is precut to fit inside the sealing gasket Use of Bio Dot SF filter paper ensures high quality results and eliminates the chance of cross well contamination 4 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 Nitrocellulose is wetted in 6x sodium sodium citrate SSC for nucleic acid applications and in Tris buffered saline TBS for protein binding Zeta Probe membrane is wetted in distilled water See Sections 9 an
37. t into solution 1 M NaHPO pH 7 2 1 M NagHPOg 7H50 FW 268 07 Dissolve 134 0 g NagHPO4 7H50 in dd H20 Add 4 ml 85 H3PO4 Adjust volume to 1 L with ddH2O 50 6 Dextran Sulfate 50 dextran sulfate 0 2 sodium azide Dissolve 50 0 g dextran sulfate and 0 2 g sodium azide in ddHoO Adjust volume to 100 ml with ddH2O Store at 4 C 50 Formamide Dilute 50 0 g formamide to 100 ml with ddH5O 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 10 ml of formamide Filter through coarse filter paper 15 For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose A Wash 2 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 2 times for 30 60 B Wash at room B Wash 2 times at 50 C in minutes at 65 C in temperature for 5 minutes 0 1x SSC 0 1 SDS 1 mM EDTA in 2x SSC 0 5 SDS 40 mM NaHPO pH 7 2 1 SDS C Wash at 68 C 2 times for 1 hour each in 0 1x SSC 0 5 SDS DNA Bound to Zeta Probe Membrane DNA Bound to Nitrocellulose 50 formamide 50 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 Section 10 Troubleshooting Guide l F
38. tein Slot Blotting 4 1 General Recommendations 1 Solution Volume The liquid in the incubation vessel should be least 0 25 cm deep to ensure the membrane is completely submerged during incubation There should be at least 0 5 ml of reagent per cm of membrane Larger volumes may be used for convenience Handling the mMembrane Wear clean plastic gloves or use forceps to avoid fingerprints on the membrane Temperature All steps are performed at room temperature 22 25 C Incubation Vessels Incubation vessels may be made of plastic or glass However since avidin binds to unsiliconized glass plastic or siliconized glass vessels should be used whenever biotin avidin systems are employed for detection Membrane Incubation Agitation with a rotating shaker platform enhances incubation efficiency If a shaker platform is not available hand mixing every few minutes and extended incubation periods will suffice Detection It is best to incubate only one membrane per vessel Should it become necessary to use more than one membrane per incubation vessel calculate the solution volume based on the membrane surface area not the vessel size 4 2 Immunoassay Procedure Detailed instructions including a comprehensive troubleshooting guide for performing immunoassays are given in the Immun Blot instruction manuals 1 Assemble the Bio Dot SF apparatus as described in Section 3 1 Prewet the membrane prior to placi
39. und to Nitrocellulose or Zeta Probe Membrane eene 10 Hybridization Protocols for RNA Probes esee 12 Probe Stripping and Rehybridization sss 13 Solutions for Protein Applications ssssssssse 13 Solutions for Nitrocellulose Membrane sssene 13 Solutions for Zeta Probe Membrane sse 14 Solutions for Nucleic Acid Applications 15 Troubleshooting Guide sssesseeee 16 References ssssssssssssssssssssseeeee nennen 19 Legal Notices eh tee ou E UR eae 19 Ordering Information ssessesssseeeee 21 Section 1 Introduction The Bio Dot SF blotting apparatus has an evenly spaced slot shaped sample template for easy slot blot sample comparisons Because the Bio Dot SF apparatus focuses the applied samples in a thin line instead of a circle this slot format makes it easy to use a densitometer to quantitate results The Bio Dot SF apparatus is provided as a complete unit or as a modular addition to the Bio Dot microfiltration system Conversion of the Bio Dot SF apparatus to the Bio Dot blotting apparatus is accomplished by purchasing the Bio Dot module which provides the 96 well sample template The Bio Dot SF slot format sample template has 48 wells with dimensions of 7 mm x 0 75 mm The wells are arranged in
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