Total RNA and Protein Isolation
Contents
1. Total RNA and Protein Isolation User manual NucleoSpin RNA Protein November 2005 Rev 01 MACHEREY NAGEL MN Total RNA and Protein Isolation Table of contents 1 Kit contents 2 Product description 2 1 2 2 2 3 2 4 2 5 The basic principle Kit specifications Handling preparation and storage of starting materials Guideline for appropriate sample amount precipitation and resolubilization volume for protein isolation Elution procedures for RNA 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protocols 5 1 5 2 5 3 5 4 Total RNA and protein purification from cultured cells and tissue with NucleoSpin RNA Protein Support protocol NucleoSpin RNA Protein Total RNA preparation from biological fluids e g serum culture medium Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 10 bacterial cells Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 5 x 10 yeast cells 6 Appendix 6 1 6 2 6 3 6 4 6 5 Quantification of protein in sample buffer Troubleshooting Literature Ordering information Product use restriction warranty MACHEREY NAGEL 11 2005 Rev 01 12 13 14 17 17 23 24 25 26 26 29 33 34 35 Total RNA and Protein Isolation 1 Kit contents Cat No Protein Precipitator PP Protein Loading Buffer PLB without re2. 11 2005 Rev 01 NucleoSpin RNA Protein 5 2 Support protocol NucleoSpin RNA Protein Total RNA preparation from biological fluids e g serum culture medium 1 Homogenization of sample Not necessary 2 Cell lysis Add 350 ul buffer RP1 to 100 ul of sample and vortex vigorously 3 Filtration of the lysate Not necessary 4 Adjust RNA binding conditions Add 350 ul of ethanol 70 to the lysate and mix by pipetting up and down approx 5 times Proceed with step 5 of the NucleoSpin RNA Protein standard protocol section 5 1 MACHEREY NAGEL 11 2005 Rev 01 23 NucleoSpin RNA Protein 5 3 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 10 bacterial cells 1 Homogenization of sample Resuspend the bacterial cell pellet Gram negative strains in 100 pl TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 containing 0 2 mg ml lysozyme by vigorous vortexing Incubate at 37 C for 10 min For preparation of RNA from Gram positive bacteria resuspend cells in 100 ul TE containing 2 mg ml lysozyme It may be necessary to optimize incubation time and lysozyme concentration depending on the bacterial strain 2 Cell lysis Add 350 ul buffer RP1 and 3 5 ul B mercaptoethanol to the suspension and vortex vigorously 3 Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filter units Place NucleoSpin Filter units i
3. 40 ul for the NucleoSpin RNA Protein kit Overall yield however will decrease when using smaller volumes For further alternative elution procedures see section 2 5 Further steps for protein purification steps 10 13 Perform sample homogenization cell lysis lysate filtration adjusting of nucleic acid binding condition and binding of nucleic acids to the NucleoSpin RNA binding column according to the NucleoSpin RNA Protein kit standard protocol steps 1 5 Use the NucleoSpin RNA Protein column flow through i e the ethanolic lysate which has been passed throught the RNA binding column and is as such deprived of nucleic acids as starting point for protein precipitation 60 pl H20 RNase free 1 min 11 000 xg 10 Protein precipitation Transfer an appropriate amount 10 700 ul of flow though into a fresh 1 5 ml microcentrifuge tube Supplied See section 2 4 as guideline for choosing an appropriate amount Add one volume of PP Protein Precipitator Mix vigorously Incubate mixture at room temperature for approximately 10 minutes Note For samples of moderate to high protein content e g 100 mg young plant leaf 30 mg liver this incubation step may be omitted For samples of low to medium protein content e g 15 mg young plant leaf the 10 min incubation increases protein yield relative to no incubation significantly An incubation of longer than one hour does not further increase pro
4. USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty MACHEREY NAGEL 11 2005 Rev 01 35 Total RNA and Protein Isolation Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact yo
5. aspirate cell culture medium and continue immediately with the addition of lysis buffer RP1 to the cell culture dish Avoid incomplete removal of the cell culture medium in order to allow full lysis activity of the lysis buffer To trypsinize adherent growing cells Aspirate cell culture medium and add and equal amount of PBS in order to wash the cells Aspirate PBS Add 0 1 0 3 trypsin in PBS and incubate for a appropriate time to detach the cells from the dish surface After cell detachment add medium transfer cells to an appropriate tube not supplied and pellet by centrifugation for 5 min at 300 x g Remove supernatant and continue with the addition of lysis buffer to the cell pellet Animal tissues are often solid and must therefore be broken up mechanically as well as lysed Depending on the disruption method the viscosity of the lysed sample has to be reduced further for optimal results It is essential for efficient RNA preparation that all the RNA contained in the sample is released from the cells by disruption and that the viscosity of the sample is reduced by homogenization The most commonly used technique for disruption of animal tissues is grinding with a pestle and mortar Grind the sample to a fine powder in the presence of liquid No Take care that the sample does not thaw during or after grinding or weighing and add the frozen powder to an appropriate aliquot of buffer RP1 containing P mercaptoethanol and mix immediat
6. directly applied onto the silica membrane during the preparation RNase free DNase is supplied with the kit Simple washing steps with two different buffers remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions with RNase free water supplied Protein is isolated from the column flow through Protein is precipitated with a special buffer Protein Precipitator PP which effectively precipitates protein After a washing step the protein pellet is dissolved in Protein Loading Buffer PLB containing the odourless reducing agent TCEP The protein can thus readily be applied to SDS PAGE analysis The RNA and protein preparation using NucleoSpin RNA Protein kits can be performed at room temperature The RNA eluate however should be treated with care because RNA is very sensitive to trace contaminations of RNases often found on general lab ware fingerprints and dust To ensure RNA stability keep RNA frozen For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 11 2005 Rev 01 5 Total RNA and Protein Isolation at 20 C for short term or 70 C for long term storage Recovered Protein dissolved in Protein Loading Buffer is unproblematic concerning stability Simultaneous Isolation of RNA Protein and DNA NucleoSpin RNA DNA buffer set The NucleoSpin RNA DNA buffer set see ordering information is a support set
7. due to residual ethanol content No problems with over drying have been observed with small sized pellets 13 Protein sample preparation Add 20 100 ul PLB Protein Loading Buffer See section 2 4 as guideline for choosing an appropriate amount Disaggregate large and visible pellets with a pipet tip to facilitate subsequent protein dissolution this is not necessary for small and invisible pellets Incubate for 3 min at 95 98 C for complete protein dissolving and denaturation Let sample cool down to room temperature MACHEREY NAGEL 11 2005 Rev 01 21 NucleoSpin RNA Protein Centrifuge for 1 min at 11 000 x g to pellet residual insolvable material Note Depending on sample amount and nature there might be no visible pellet of insolvable material up to large pellets of different size and structure Do not disturb precipitates at this stage Protein will be in the supernatant Do not centrifuge samples cooled down to 0 4 C SDS may precipitate at this temperature Recover supernatant for further analysis Note At this stage samples can be stored at 20 C 4 C for several month days After storage equilibrate sample to room temperature mix and then centrifuge briefly before withdrawal of sample aliquots Repeated sample denaturing for 3 min at 95 98 C is not necessary Repetitive withdrawal freezing and thawing for at least three times has shown constant sample quality 22 MACHEREY NAGEL
8. for RNA and DNA isolation in conjunction with NucleoSpin RNA II NucleoSpin RNA Plant or NucleoSpin RNA Protein This patent pending technology enables successive elution of DNA and RNA from one NucleoSpin column with low salt buffer and water respectively DNA and RNA are immediately ready for downstream applications The combination with NucleoSpin RNA Protein allows parallel isolation of RNA DNA and Protein from one undivided sample 2 2 Kit specifications e NucleoSpin RNA Protein kits are recommended for the isolation of total RNA and protein from cultured cells and tissue The NucleoSpin RNA Protein kits allow purification of pure RNA with an Ageo280 ratio generally exceeding 1 9 measured in TE buffer pH 7 5 e The isolated RNA is ready to use for applications like reverse transcriptase PCR RT PCR primer extension or RNase protection assays e Integrity of RNA isolated from e g eukaryotic cells is examined by denaturing agarose gel electrophoresis rRNA bands are sharp with the 28S band being about twice as intense as the 18S band see Figure 1 Figure 1 Total RNA from mouse liver was isolated with a NucleoSpin RNA kit and separated on a 1 2 formaldehyde agarose gel e The isolated protein is ready to use for SDS PAGE and Western Blot analysis 6 MACHEREY NAGEL 11 2005 Rev 01 Protein is easily visualized by SDS PAGE M A Total RNA and Protein Isolation Fig 2 The protein
9. in order to avoid sedimentation of any precipitate Bind RNA For each preparation take one NucleoSpin RNA Protein column light blue placed in a 2 ml centrifuge tube and load the lysate Centrifuge for 30 s at 11 000 x g Place the column in a new collecting tube RNA and DNA are bond to the column matrix protein load lysate is contained in the flow through Maximal loading capacity of NucleoSpin RNA Protein columns is 750 ul Repeat the procedure if larger volumes are to be processed 30s 11 000 x g For RNA isolation continue with step 6 It is recommended first to continue RNA isolation protocol and do protein purification subsequently For protein isolation recover flow through and continue with step 10 The protein containing flow through is stable for several hours at 4 8 C Further steps for RNA purification steps 6 9 6 Desalt silica membrane 350 ul MDB Add 350 ul MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 1 min to dry the membrane Salt removal will make the following DNase I digest much more j effective If the column outlet has come into contact with the C3 1 min flow through for any reason discard the flow through and 11 000 x g centrifuge again for 30 s at 11 000 x g 18 MACHEREY NAGEL 11 2005 Rev 01 Digest DNA Prepare DNase reaction mixture in a sterile microcentrifuge tube for each isolation add 10 ul reconstituted DNase I also
10. see section 3 to 90 pl DNase reaction buffer Mix by flicking the tube Apply 95 ul DNase reaction mixture directly onto the center of the silica membrane of the column Incubate at room temperature for 15 min Wash and Dry silica membrane 1 wash Add 200 ul buffer RA2 to the NucleoSpin RNA Protein column Centrifuge for 30 s at 11 000 x g Place the column into a new collecting tube Buffer RA2 will inactivate the DNase 2 wash Add 600 ul buffer RA3 to the NucleoSpin RNA Protein column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collecting tube 3 wash Add 250 ul buffer RA3 to the NucleoSpin RNA PROTEIN column Centrifuge for 2 min at 11 000 x g to dry the membrane completely Place the column into a nuclease free 1 5 ml microcentrifuge tube supplied If for any reason the liquid level in the collecting tube has reached the NucleoSpin RNA Protein column after centrifugation discard flow through and centrifuge again MACHEREY NAGEL 11 2005 Rev 01 NucleoSpin RNA Protein 95 ul DNase reaction mixture RT 15 min 200 pl RA2 30 s 11 000 x g 600 ul RA3 30 s 11 000 x g 250 pl RA3 2 min 11 000 x g 19 NucleoSpin RNA Protein Elute highly pure RNA Elute the RNA in 60 pl H20 RNase free supplied and centrifuge at 11 000 x g for 1 min If higher RNA concentrations are desired elution can be done with
11. through immediately after loading Unclear results with commonly used protein quantification systems Most commonly used protein quantification systems are incompatible with concentrations of SDS and or reducing agents present in Protein Loading Buffer e Use quantification method as described in section 6 1 which is compatible with Protein Loading Buffer PLB 32 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation Problem Possible cause and suggestions A small sample amount was used and or a small volume of column flow through was used for precipitation No protein precipitate e Formation of a visible protein pellet is not required for sufficient pellet visible protein recovery Even invisible protein pellets commonly yield enough protein for SDS PAGE and Western Blot analysis 6 3 Literature Coombs LM Pigott D Proctor A Eydmann M Denner J and Knowles MA 1990 Simultaneous isolation of DNA RNA and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiodyanate Analytical Biochemistry 188 pp338 343 Banerjee S Smallwood A Chambers AE and Nicolaides K 2003 Quantitative recovery of immunoreactive proteins from clinical samples following RNA and DNA isolation BioTechniques 35 3 pp 450 456 Getz EB Xiao M Chakrabarty T Cooke R and Selvin PR 1999 A comparison between the sulfhydryl reductants Tris 2 carboxyethyl phosphine and Dith
12. 20 21 22 S 13 thiocyanate skin and if swallowed MDB guanidine Substance does not have to be specially labeled as hazardous thiocyanate lt 10 Reducing Tris 2 Causes burns R 34 S 26 27 agent TCEP carboxyethyl xi 36 37 39 phosphine Hydrochloride Risk Phrases R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed R 34 R 42 43 Causes burns May cause sensitization by inhalation and skin contact Safety Phrases Keep away from food drink and animal feedstuffs Do not breathe dust Avoid contact with the skin S 13 S 22 S 24 S 26 S27 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Take off immediately all contaminated clothing S 36 37 39 Wear suitable protective clothing gloves and eye face protection Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 MACHEREY NAGEL 11 2005 Rev 01 16 NucleoSpin RNA Protein 5 Protocols 5 1 Total RNA and protein purification from cultured cells and tissue with NucleoSpin RNA Protein Joint protocol steps for RNA and protein purification 1 Homogenization of sample Disrupt up to 30 mg of tissue for homogenization disrupt methods see section 2 4 sample Up to 5 x 10 eukaryotic cultured cells are collected by centrifugation and lysed by addition of RP1 directly 2 Cell
13. AGEL 11 2005 Rev 01 31 Total RNA and Protein Isolation Problem Possible cause and suggestions Carryover of ethanol or salt e Do not let the flow through touch the column outlet after the second RA3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic buffer RA3 completely Suboptimal Check if buffer RA3 has been equilibrated to room temperature performance of before use Washing at lower temperatures lowers efficiency of RNA in salt removal by RAS downstream experiments Store isolated RNA properly e Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C Protein pellets exceeding several millimeters in size are hard to trouble with dissolve resel Bilizatlon Use smaller volumes of column flow through for protein of precipitated Sate a can RR precipitation in order to obtain small sized pellets Even protein in PLB Veen 2 TCEP invisible protein pellets commonly yield enough protein for SDS PAGE and Western Blot analysis Protein Protein pellet has not been dried sufficiently and contains residual dissolved in ethanol er B ee S e Increase drying time or decrease pellet size by precipitating a ll f PAGE gel slot smaller volume of column flow
14. MACHEREY NAGEL 11 2005 Rev 01 Problem Total RNA and Protein Isolation Possible cause and suggestions Contamination of RNA with genomic DNA DNase not active e Reconstitute and store lyophilized DNase according to instructions given in section 3 DNase solution not properly applied e Pipette DNase solution directly onto the center of the silica membrane Too much cell material used e Reduce quantity of cells or tissue used DNA detection system too sensitive e The amount of DNA contamination is significantly reduced during the on column DNase digestion Anyhow we can not guarantee that the purified RNA is 100 free of DNA therefore in very sensitive applications it might be possible to detect DNA The NucleoSpin RNA Protein II Plant system is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1kb fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the DNase is applied however a strong PCR fragment is obtained if DNase is omitted The eventuality of DNA detection with PCR increases with the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt plasmid transfected into cells decreasing of PCR amplicon size Use larger PCR targets e g gt 500bp or intron spanning primers if possible MACHEREY N
15. P1 lysate Check that 50 ethanol is available as additional solution to wash the protein pellet Before starting any NucleoSpin RNA Protein protocol prepare and consider the following RNase free DNase I Add indicated volume of RNase free water see table below to the DNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the DNase Be careful not to mix DNase vigorously as DNase is sensitive to mechanical agitation Dispense into aliquots and store at 18 C The frozen working solution is stable for 6 months Do not freeze thaw the aliquots more than three times Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to the RA3 concentrate Store buffer RA3 at room temperature 20 25 C for up to one year Protein Loading Buffer PLB and reducing agent TCEP For SDS PAGE under reducing conditions transfer PLB without reducing agent to the lyophilized reducing agent TCEP Mix well until the reducing agent is dissolved completely this process will require several minutes Protein Loading Buffer containing reducing agent TCEP PLB TCEP is stable for several days at room temperature 18 25 C and several month at 4 C For long term storage of PLB TCEP keep at 20 C For 50 and 250 prep kits For better handling PLB TCEP may be transferred into the original PLB vial with screw cap 14 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Pro
16. amount loaded per lane corresponds to 14 000 HeLa cells A 0 43 mg liver B and1 43 mg garden cress seedling C respectively Table 1 Kit specifications at a glance NucleoSpin RNA Protein Sample size RNA yield Protein yield Elution volume RNA Resolubilization volume Protein Binding capacity RNA Time prep RNA Time prep protein Spin column type up to 5 x 10 cells up to 30 mg tissue up to 70 ug up to 1200 ug 40 120 ul 10 100 ul 100 ug lt 30 min 6 preps 35 min 6 preps mini MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation The standard protocol section 5 1 allows the purification of up to 70 ug of total RNA per NucleoSpin RNA Protein column from up to 5 x 10 cultured cells or 30 mg of tissue also see table 1 The isolated RNA can be used as template in a RT PCR reaction Generally 1 10 of the eluate of total RNA prepared from 1 x 10 cells or 10 mg of tissue is sufficient as template for RT PCR If possible intron spanning primers should be used for RT PCR The RNA prepared from such high amounts is generally free of residual DNA although minute traces of DNA may remain in the preparation if large amounts of material rich in nucleic acids are used However if the isolated RNA will be used as template in a RT PCR reaction we recommend that lower quantities of sample be used depending on cell or tissue type in the range of 1 x 10 cultured cells
17. and RNA are isolated without splitting the sample prior to protein RNA extraction Thus protein and RNA are obtained from one and the same sample and not from two similar portions of one sample This is especially valuable for unique small and precious samples Isolated RNA is suitable for all common downstream applications RNA isolated with the NucleoSpin RNA Protein Kit is of identical quality as RNA isolated with the well proven NucleoSpin RNA II Kit Isolated protein is immediately suitable for SDS PAGE and Western blot analysis RNA and Protein Isolation One of the most important aspects in the isolation of RNA and Protein is to prevent their degradation during the isolation procedure With the NucleoSpin RNA Protein method cells are lysed by incubation in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates virtually all enzymes e g RNases and proteases which are present in almost all biological materials The buffer dissolves even hardly soluble protein creates appropriate binding conditions which favor adsorption of RNA to the silica membrane and enables protein to pass the specially treated NucleoSpin RNA Protein column virtually quantitatively Expensive and harmful proteinase inhibitors or inhibitor cocktails are not necessary due to the denaturing properties of the lysis buffer Contaminating DNA which is also bound to the silica membrane is removed by a DNase solution which is
18. btained with the NucleoSpin RNA Protein kit are virtually free of nucleic acids thus protein quantification is not affected Upon addition of TCA Trichloracetic acid to the sample protein precipitates and causes turbidity The degree of turbidity is used for quantification relative to a sample with known protein concentration This test enables determination of protein concentration in the range 5 ng ul 20 ug ul by using variable sample volumes of 1 60 ul Recommended sample volume For protein concentration in Protein dissolved in PLB TCEP the range of 60 yl 0 005 0 33 pg pl 20 ul 0 015 1 0 ug ul 1 ul 0 3 20 ug ul Material TCA 60 Trichloracetic acid not supplied Protein Loading Buffer with reducing agent PLB TCEP BSA Bovine Serum Albumin not supplied Multititer plate not supplied Composition of PLB TCEP 125 mM BisTris Bis 2 hydroxyehtyl imino tris hydroxymethyl methane 10 SDS sodium dodecyl sulphate 50 mM TCEP Tris 2 carboxyethyl phosphine Hydrochloride 20 glycerol 0 02 brome phenol blue pH 6 8 26 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation Method Prepare a BSA stock solution with 40 mg ml BSA in H20 Prepare a BSA dilution series BSA solution PLB TCEP __ BSA in 20 ul 1 awa stock solution 97 5 ul 1 ug yl 20 ug 2 50 ul from 1 50 ul 0 5 ug ul 10 ug 3 50 ul from 2 50 ul 0 25 ug
19. cause and suggestions Poor RNA quality or yield continued lonic strength and pH influence Aso absorption as well as ratio A 260 280 For adsorption measurement use 5 mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of A260 A280 ratios for measurement of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Biotechniques 22 474 481 Sample material Sample material not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liquid N2 Samples should always be kept at 70 C Never allow tissues to thaw before addition of buffer RP1 Perform disruption of samples in liquid No Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filter Filter L units for easy homogenization of disrupted starting material Clogged NucleoSpin column Poor RNA quality or yield 30 Sample material Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material or use larger volume of RP1 Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filter Filter L units for easy homogenization of disrupted starting material
20. ducing agent Reducing agent TCEP Buffer RP1 Buffer RA2 Buffer RA3 concentrate Buffer MDB Membrane Desalting Buffer DNase reaction buffer DNase RNase free Iyophilized H20 RNase free NucleoSpin Filter units violet ring NucleoSpin RNA Protein columns light blue ring plus collecting tube NucleoSpin collecting tubes 1 5 ml microcentrifuge tubes Protocol NucleoSpin RNA Protein 10 preps 740933 10 9 ml 2x 1ml 2x 14mg 10 ml 15 ml 5 ml 10 ml 3 ml 1 vial 5 ml 10 10 30 20 50 preps 740933 50 45 ml 7 5 ml 107 mg 25 ml 15 ml 12 5 ml 25 ml 7 ml 1 vial 15 ml 50 50 150 100 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 11 2005 Rev 01 250 preps 740933 250 225 ml 5x 7 5 ml 5 x 107 mg 125 ml 80 ml 75 ml 125 ml 35 ml 5 vials 65 ml 250 250 750 500 1 Total RNA and Protein Isolation 2 Product description 2 1 The basic principle Introduction Studies of gene expression at the level of transcription and translation by quantification of RNA and protein are often hampered by the small sample size and the necessity of different often incompatible techniques for RNA and protein isolation Samples may comprise biopsies tumors tissues transgene organisms and others The NucleoSpin RNA Protein Kit however enables isolation of RNA and protein from diverse sample types Protein
21. ely The broken up tissue must then be homogenized with a NucleoSpin Filter Filter L unit or by passing gt 5 through a 0 9 mm syringe needle Thawing of undisrupted animal tissue should be exclusively done in the presence of buffer RP1 during simultaneous mechanical disruption e g with a rotor stator homogenizer This ensures that the RNA is not degraded by RNases before the 10 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation preparation has started The spinning rotor disrupts and simultaneously homogenizes the sample by mechanical shearing of DNA within seconds up to minutes homogenization time depends on sample Take care to keep the rotor tip submerged in order to avoid excess foaming To degenerate evolved foam centrifuge 1 min at 400 x g Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes Bacteria and yeasts have to be incubated in lysozyme or lyticase zymolase solutions respectively see support protocols in section 4 By this treatment the robust cell walls of these organisms are digested or at least weakened which is essential for effective cell lysis by buffer RP1 For microorganisms with extremely resistant cell walls like some Gram positive bacterial strains it may be necessary to optimize the conditions of the treatment with lytic enzymes or the cultivation conditions After lysis homogenization is achieved by the use of a N
22. ion of only a portion of the column flow through e g 100 ul is recommended and will yield enough protein in terms of absolute amount and concentration for SDS PAGE Western analysis Sample type and amount Protein yield cultured human cells e g HeLa approx 10 cells 50 150 ug plants e g garden cress approx 100 mg 150 350 ug animal tissue e g pig liver approx 30 mg 500 1200 ug MACHEREY NAGEL 11 2005 Rev 01 9 Total RNA and Protein Isolation 2 3 Handling preparation and storage of starting materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are flash frozen in liquid N gt immediately and stored at 70 C or processed as soon as possible Samples can be stored in lysis buffer RP1 after disruption at 70 C for up to one year at 4 C for up to 24 hours or up to several hours at room temperature Frozen samples are stable up to 6 months Frozen samples in buffer RP1 should be thawed slowly before starting with the isolation of total RNA Wear gloves at all times during the preparation Change gloves frequently Cultured animal cells are collected by centrifugation and directly lysed by adding buffer RP1 according to step 2 of the standard protocol see section 5 1 Cell lysis of adherent growing cells in a culture dish Completely
23. iothreitol for use in protein biochemistry Analytical Biochemistry 273 73 80 Hoemann CD Sun J Chrzanowski V and Buschmann MD 2002 A multivalent assay to detect glycosaminoglycan protein collagen RNA and DNA content in milligam samples of cartilage or hydrogel based repair cartilage Analytical Biochemistry 300 1 10 Karlsson JO Ostwald K Kabjorn C and Andersson M 1994 A method for protein assay in Laemmli buffer Analytical Biochemistry 219 144 146 MACHEREY NAGEL 11 2005 Rev 01 33 Total RNA and Protein Isolation 6 4 Ordering information Product Cat No Pack of NucleoSpin RNA II 740955 10 10 NucleoSpin RNA II 740955 20 20 NucleoSpin RNA II 740955 50 50 NucleoSpin RNA II 740955 250 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Clean up 740948 10 10 NucleoSpin RNA Clean up 740948 50 50 NucleoSpin RNA Clean up 740948 250 250 NucleoSpin RNA DNA buffer set 740944 100 DNase set 740963 1 set NucleoSpin Filter 740606 50 NucleoSpin collection tubes 740600 1000 Porablot see price list Blotting paper see price list 34 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation 6 5 Product use restriction warranty NucleoSpin RNA Protein kits components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic p
24. lysis 350 pl RP1 Add 350 pl buffer RP1 and 3 5 ul B mercaptoethanol to 3 5 ul B me the cell pellet or to ground tissue and vortex vigorously 3 Filtration of the lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter units Place NucleoSpin Filter units violet in a collecting tube apply the mixture and centrifuge for 1 min at 11 000 x g The lysate may be passed alternatively gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe 6 4 min 11 000 xg In case of visible pellet formation depending on sample amount and nature transfer supernatant without any ado formed pellet to a new 2 ml centrifuge tube not included Important To process higher amounts of cells gt 1 x 10 or tissue gt 10 mg the lysate should first be homo genized using the 0 9 mm needle 20 gauge followed by filtration through NucleoSpin filter units MACHEREY NAGEL 11 2005 Rev 01 17 NucleoSpin RNA Protein Adjust RNA binding conditions Discard the NucleoSpin Filter unit and add 350 wl ethanol 70 to the homogenized lysate and mix by 350 pl pipetting up and down approx 5 times 70 EtOH After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to mix disaggregate any precipitate by mixing and to load all of the disaggregated precipitate on the column as described in step 5 Do not centrifuge at this stage
25. n collecting tubes apply mixture and centrifuge for 1 min at 11 000 x 9 In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 2 ml centrifuge tube not included Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe 4 Adjust RNA binding conditions Add 350 ul of ethanol 70 and mix by pipetting up and down approx 5 times Proceed with step 5 of the NucleoSpin RNA Protein standard protocol section 5 1 Because of the much greater concentration of genome equivalents in a nucleic acid preparation of bacteria compared with eukaryotic material it may be necessary to use a lower quantity of cells for the preparation 24 MACHEREY NAGEL 11 2005 Rev 01 NucleoSpin RNA Protein 5 4 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 5 x 10 yeast cells 1 Homogenization of sample Harvest 2 5 ml of YPD culture 5 000 x g 10 min Resuspend pellet in sorbitol lyticase buffer 50 100 U lyticase or zymolase in 1 M sorbitol 100 mM EDTA and incubate at 30 C for 30 min Pellet the resulting spheroplasts by centrifugation 1 000 x g 10 min It may be necessary to optimize incubation time and lyticase zymolase concentration depending on the yeast strain 2 Cell lysis Add 350 ul buffer RP1 and 3 5 yl B mercaptoethanol to the suspension and vortex vigorousl
26. ng Buffer PLB 28 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation 6 2 Troubleshooting Problem Possible cause and suggestions RNase contamination RNA is e Create an RNase free working environment Wear gloves during all steps of the procedure Change gloves frequently degraded no RNA Use of sterile disposable polypropylene tubes is l l obiained recommended Keep tubes closed whenever possible during the preparation Glassware should be oven baked for at least 2 hours at 250 C before use Reagents not applied or restored properly e Reagents not properly restored Add the indicated volume of nuclease free water to DNase vial and 96 ethanol to buffer concentrate RA3 and mix Reconstitute and store lyophilized DNase according to instructions given in section 3 e Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added Poor RNA e No ethanol has been added after lysis Binding of RNA to the quality silica membrane is only effective in the presence of ethanol or yield Kit storage Reconstitute and store lyophilized DNase according to instructions given in section 3 Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination MACHEREY NAGEL 11 2005 Rev 01 29 Problem Total RNA and Protein Isolation Possible
27. on 2 5 Elution procedures for RNA It is possible to adapt elution method and volume of water used for the subsequent application of interest In addition to the standard method described in the individual protocols recovery rate about 70 90 there are several modifications possible e High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acid will be eluted e High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for reelution Eluted RNA should immediately be put and always kept on ice for optimal stability because almost omnipresent RNases general lab ware fingerprints dust will degrade RNA For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 11 2005 Rev 01 13 Total RNA and Protein Isolation 3 Storage conditions and preparation of working solutions Attention Buffers RP1 RA2 and MDB contain guanidine thiocyanate Wear gloves and goggles Store lyophilized RNase free DNase I at 4 C on arrival stable up to 1 year Store lyophilized reducing agent TCEP at 4 C on arrival All other kit components should be stored at room temperature 20 25 C and are stable up to one year Storage at lower temperatures may cause precipitation of salts Check that 70 ethanol is available as additional solution to adjust binding conditions in the R
28. or 10 mg of tissue resulting in about 20 yg of RNA The kit can be used for preparing RNA from different amounts of sample material according to the following table Sample Amount Cultured animal cells up to 5 x 10 e g HeLa cells Animal tissue up to 30 mg Bacteria up to 1 x 10 Yeast up to 5 x 10 Depending on sample type the average yield is around 5 ug 70 ug total RNA see Table 2 The Azgoy2go ratio indicating purity of the RNA generally exceeds 1 9 Table 2 Overview on average yields of total RNA isolation using NucleoSpin RNA Protein Sample average yield yg 8 x 104 HeLa cells 1 5 4 x 10 HeLa cells 4 1 x 10 HeLa cells 14 2 x 10 HeLa cells 21 2 5 x 10 HeLa cells 25 5 x 10 HeLa cells 50 MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolation Protein yield Protein yield depends on sample type amount and quality as well as on homogenization efficiency Further the utilized quantification method influences determined protein yield The following values were determined according to the method described in appendix section 6 1 and shall serve as a guideline for expected protein yield It is assumed that the complete sample amount is processed i e the complete lysed sample is after ethanol addition loaded onto the column and the complete 700 ul flow through is subjected to protein precipitation In many cases precipitat
29. rognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin RNA Protein kits for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR
30. tein Isolation e If SDS PAGE at non reducing conditions is intended consider the following A Omit addition the reducing agent TCEP to buffer PLB B Omit addition of B mercaptoethanol to lysis buffer RP1 e If other reducing agents than TCEP are preferred e g DTT P mercaptoethanol appropriate amounts should be added to PLB Please consider limited stability of DTT compared to TCEP NucleoSpin RNA Protein 10 preps 50 preps 250 preps Cat No 740933 10 740933 50 740933 250 Buffer RA3 5 ml 12 5 ml 3 x 25 ml concentrate add 20 ml ethanol add 50 ml ethanol add to each vial 100 ml ethanol DNase 1 vial 1 vial 5 vials RNase free add 230 ul add 540 ul add to each vial lyophilized RNase free water RNase free water 540 ul RNase free water MACHEREY NAGEL 11 2005 Rev 01 15 Total RNA and Protein Isolation 4 Safety instructions risk and safety phrases The following components of the NucleoSpin RNA Protein kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety Contents Symbol Phrases Phrases DNase DNase x Xn May cause sensitization by inhalation R 42 43 S 22 24 lyophilized and skin contact Buffer RP1 guanidine x Xn Harmful by inhalation in contact with R 20 21 22 S 13 thiocyanate skin and if swallowed Buffer RA2 guanidine x xn Harmful by inhalation in contact with R
31. tein yield Centrifuge for 5 min at 11 000 x g 20 MACHEREY NAGEL 11 2005 Rev 01 NucleoSpin RNA Protein 11 Protein wash Remove supernatant by pipetting or decanting as complete as possible Add 500 ul of 50 ethanol to the pellet mixing or incubation at this step is not necessary Centrifuge 1 min at 11 000 x g Remove supernatant by pipetting or decanting as completely as possible Note Protein precipitate at this stage is quite different in appearance depending on kind and amount of starting material The appearance might be no visible pellet or precipitate e g for 10 000 cell 0 3 mg liver and 1 mg leaf samples a greenish tube wall coating on one side of the tube for e g leaf material green or white pellet at the bottom of the tube e g for leaf and liver samples respectively green or white crumbs at one side of the inner wall of the centrifuge tube e g for leaf and liver samples respectively If no precipitate is visible mark the side of the tube where a precipitate is expected in order to avoid touching this side of the inner tube wall with the pipet tip during the washing step 12 Dry protein pellet Dry precipitate for 5 10 min at room temperature keep lid open Note Large pellets e g complete precipitation of 700 ul column flow through form a 30 mg liver sample need longer drying duration Samples which are dried incomplete my cause problems when loading the sample onto the gel
32. ucleoSpin Filter units or the syringe needle method MACHEREY NAGEL 11 2005 Rev 01 11 Total RNA and Protein Isolation 2 4 Guideline for appropriate sample amount precipitation and resolubilization volume for protein isolation The following table shall serve as a first guide for choosing appropriate amounts of sample material precipitation and resolubilization volume Depending on sample type and downstream application Coomassie or silver stain sensitivity of antibody detection system appropriate volumes might deviate from the table below and have to be determined experimentally amount of cultivated cells e g HeLa animal tissue e g liver plant tissue e g garden cress leaf sample 10 10 104 30mg 3mg 0 3mg 100mg 10mg 1mg lysis buffer RP1 350 ul ethanol 350 ul column flow through to be precipitated 35 ul 350 ul 700 ul 35 ul 350 ul 700 ul 35 ul 350 ul 700 ul sample buffer PLB used for protein pellet solubilisation 100 ul 100 ul 20 ul 100 ul 100 ul 20 ul 100 ul 100 ul 20 ul protein sample to be analysed on SDS PAGE with coomassie stain 10 pl protein sample to be analysed on SDS PAGE with silver stain 1 ul protein sample analysed on western blot 1 10 ul MACHEREY NAGEL 11 2005 Rev 01 Total RNA and Protein Isolati
33. ul 5 ug 4 50 ul from 3 50 ul 0 125 ug ul 2 5 Ug 5 50 ul from 4 50 ul 0 063 ug ul 1 25 ug 6 50 ul from 5 50 ul 0 031 ug ul 0 625 ug 7 50 ul from 6 50 ul 0 016 yg ul 0 312 ug 8 50 ul 0 ug l 0 ug The prepared BSA dilution series is sufficient for subsequent determination of two calibration curves 1 Add 20 ul of each dilution series sample 1 8 in microtiter plates wells Add 20 ul of samples protein dissolved in PLB TCEP with unknown protein concentration to further wells alternatively 1 60 ul Add 40 ul PLB TCEP to each well Final volume 60 ul alternatively add 0 55 ul if other volumes than 20 ul of sample are used in step 2 Add 40 ul TCA 60 to each well Mix until complete colour change from blue to yellow Incubate for 30 min 3 min at room temperature Measure absorbance at 570 nm Determine protein concentration of samples in relation to dilution series Measurement of absorption in the range of 530 700 nm is suitable and will typically result in correlation coefficients of 2 0 99 concentration of BSA dilution series vs obtained absorption values MACHEREY NAGEL 11 2005 Rev 01 27 Total RNA and Protein Isolation Quantification of protein in Protein Loading Buffer PLB 1 2 gs 0 1 4 E E 2 N 0 01 0 001 i j 0 1 10 100 BSA amount per well yg Fig 3 BSA standard curve for determination of protein in Protein Loadi
34. ur local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 and 275 e mail TECH BIO mn net com 36 MACHEREY NAGEL 11 2005 Rev 01
35. y 3 Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filter units Place NucleoSpin Filter units in collecting tubes apply mixture and centrifuge for 1 min at 11 000 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 2 ml centrifuge tube not included Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe Proceed with step 4 of the NucleoSpin RNA Protein standard protocol section 5 1 Due to the much higher concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material it may be necessary to use a lower quantity of cells for the preparation MACHEREY NAGEL 11 2005 Rev 01 25 NucleoSpin RNA L 6 Appendix 6 1 Quantification of protein in sample buffer Quantification of protein in the sample buffer is occasionally helpful prior to SDS PAGE and Western Blot analysis However major protein quantification assays are influenced by incompatible with SDS reducing agents commonly present in protein sample buffers for SDS PAGE The procedure presented below based on the publication of Karlsson et al 1994 is suitable for quantification of protein in Protein Loading Buffer PLB Nucleic acids disturb protein quantification as described by Karlsson Protein samples o
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