Total RNA and Protein Isolation
Contents
1. 3 Filtration of the lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter Place NucleoSpin Filter violet ring in a Collection Tube apply the mixture and centrifuge for 1 min at 11 000 x g j 1 min The lysate may be passed alternatively 5 times through a 11 000 x g 0 9mm needle 20 gauge fitted to a syringe 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 includ ed 18 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein Important To process higher amounts of cells gt 1 x 10 or tissue gt 10 mg the lysate should first be homogenized using the 0 9 mm needle 20 gauge followed by filtration through NucleoSpin Filter Adjust RNA binding conditions Discard the NucleoSpin Filter and add 350 pl ethanol 70 to the homogenized lysate and mix by pipetting up and down approx 5 times Alternatively transfer flow through into a new 1 5 ml microcentrifuge tube not provided add 350 ul ethanol 70 and mix by vortexing 2 x 5 s After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to dis aggregate any precipitate by mixing and to load all of the dis aggregated precipitate on the column as described in step 5 Do not centrifuge at this stage in order to avoid sedimenta tion of2. Sample Amount Cultured animal cells e g HeLa cells MPO NG Animal tissue Up to 30 mg Bacteria Up to 1 x 10 Yeast Up to 5 x 107 e Depending on sample type the average yield is around 5 ug 70 yg total RNA see table 3 The A A ratio indicating purity of the RNA generally exceeds 1 9 Table 3 Overview on average yields of total RNA isolation using NucleoSpin RNA Protein Sample Average yield 8 x 104 HeLa cells 1 5 ug 4 x 10 HeLa cells 4 ug 1 x 10 HeLa cells 14 ug 2 x 10 HeLa cells 21 ug 2 5 x 10 HeLa cells 25 ug 5 x 10 HeLa cells 50 ug MACHEREY NAGEL 10 2008 Rev 05 9 Total RNA and Protein Isolation Protein yield Protein yield depends on sample type amount and quality as well as on homoge nization efficiency Further the utilized quantification method influences determined protein yield The following values were determined with the MACHEREY NAGEL Protein Quantification Assay 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 after ethanol addition is loaded onto the column and the complete 700 ul flow through is subjected to protein precipitation Note that in many cases precipitation of only a portion of the column flow through e g 100 yl is recommended and will yield enough protein in terms of absolute amount and concentration for SDS PAGE and Western Blot analysis
3. 10 x 40 ul 50 x 40 ul 250 x 40 ul see page 38 step 3 1 85 ml 7 45 ml 37 25 ml Total 2 ml supplied 7 5 ml supplied 5x7 5 ml supplied 36 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation Additional volumes of PSB TCEP are required if 1 protein pellets are dissolved in more than 100 ul higher volume required for solubilization of protein 2 less than 20 ul of protein is quantified higher volume required to fill up the pro tein samples to 60 ul for quantification 3 additional BSA dilution series have to be generated for additional calibration curves For ordering information please see section 6 4 Method Prepare a BSA stock solution with 40 mg ml BSA in H O Prepare a BSA dilution series Tube Add PSB Add BSA solution Resulting BSA Resulting BSA to tube to tube concentration in 20 ul 1 97 5 ul 2 5 ul BSA stock solution 1 yg ul 20 ug 40 mg ml 2 50 ul 50 ul from tube 1 0 5 pg pl 10 ug 3 50 ul 50 ul from tube 2 0 25 yg ul 5 ug 4 50 ul 50 ul from tube 3 0 125 pg pl 2 5 ug 5 50 ul 50 ul from tube 4 0 063 pg pl 1 25 ug 6 50 ul 50 ul from tube 5 0 031 pg pl 0 625 ug 7 50 ul 0 ug ul 0 ug Make sure that the protein concentration of your sample lies within the range of the largest 1 and smallest 6 value of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range The prepared BSA dilution series
4. As a guideline for appropriate precipitation volumes see section 2 4 Table 4 Typical protein yield Sample type and amount Protein yield au a e a 50 150 pg e g garden a ne 100 mg 150 350 ug e g pig ver approx 30mg __ 700 1200 ng 10 MACHEREY NAGEL 10 2008 Rev 05 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 dis rupted in the presence of RNase inhibiting or denaturing agents Therefore it is impor tant that samples are flash frozen in liquid N immediately and stored at 70 C or pro cessed 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 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 cul
5. 5 5 Support protocol NucleoSpin RNA Protein Total RNA preparation from RNAl ater treated samples Before starting the preparation e Check if Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 1 Sample preparation Remove RNA ate solution Cut an appropriate amount of tissue 2 Cell lysis Add 350 pl Buffer RP1 and 3 5 pl B mercaptoethanol B ME to the sample Disrupt the sample material by using e g rotor stated homogenizers for ho mogenization methods see section 2 3 As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RP1 Proceed with step 3 of the NucleoSpin RNA Protein standard protocol sec tion 5 1 28 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein 5 6 Support protocol NucleoSpin RNA Protein rDNase digestion in solution The on column rDNase digestion in the standard protocol is very efficient and resulting in minimal residual DNA content of the purified RNA This DNA will not be detectable in most downstream applications Despite this there are still certain applications which require even lower contents of residual DNA However removal of DNA to a completely undetectable level is challenging and the efficiency of an on column DNA digestion is sometimes not sufficient for downstream applications requiring lowest residual content of DNA Atypical exa
6. Total RNA and Protein Isolation User Manual NucleoSpin RNA Protein October 2008 Rev 05 MACHEREY NAGEL MN Total RNA and Protein Isolation Protocol at a glance Rev 05 NucleoSpin RNA Protein 1 Homogenization of sample e 30 mg 2 Cell lysis 350 ul RP1 3 5 ul B mercapto ethanol 3 Filtration of S lysate 1 min H 11 000 x g GER AES 350 ul ethanol 70 conditions 5 Bind RNA oS wa 11 000 x g Protein Purification protein in the column flow through RNA Purification RNA bound to the silica membrane 6 Desalt silica membrane 350 ul MDB 1 min 11 000 x g 7 Digest 95 ul DNase DNA reaction mixture RT 15 min 8 Wash and dry silica 1 wash 200 pl RA2 membrane 2 wash 600 ul RA3 30s 11 000 x g 37 wash 200 pl RA2 2 min 11 000 x g 9 Elute highly 60 ul H O pale RNs RNase free ed 1 min 11 000 x g MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com 10 Precipitate 10 700 ul protein flow through 1 vol PP RT 10 min eS 5 min 11 000 x g 11 Wash protein 500 ul ethanol 50 1 min 11 000 x g 12 Dry protein RT pellet 5 10 min 13 Prepare 20 100 ul protein PLB TCEP sample 3 min 95 98 C cS 1 min 11 000 x g Total RNA and Protein Isolation Table of contents 1 Components 4 1 1 Kit contents 4
7. without reducing agent Reducing Agent TCEP Lysis Buffer RP1 Wash Buffer RA2 Wash Buffer RA3 Concentrate Membrane Desalting Buffer MDB Reaction Buffer for rDNase rDNase RNase free lyophilized RNase free H O NucleoSpin Filters violet rings NucleoSpin RNA Protein Columns light blue rings plus Collection Tubes Collection Tubes 2 ml Collection Tubes 1 5 ml User Manual 10 preps 740933 10 9ml 2x1 ml 2x14 mg 10 ml 15 ml 5 ml 10 ml 3 ml 1 vial size C 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 size D 15 ml 50 50 150 100 For preparation of working solutions and storage conditions see section 3 250 preps 740933 250 225 ml 5x 7 5 ml 5 x 107 mg 125 ml 80 ml 3 x 25 ml 125 ml 35 ml 5 vials size D 65 ml 250 250 750 500 4 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol to prepare Wash Buffer RA3 e 70 ethanol to adjust RNA binding conditions e 50 ethanol to wash protein pellet e Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane to supplement lysis buffer Consumables e 1 5 ml microcentrifuge tubes for sample lysis e Disposab
8. during all steps of the procedure Change gloves frequently no RNA Use of sterile disposable polypropylene tubes is recommend obtained ed Keep tubes closed whenever possible during the prepara tion 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 RNase free water to rDNase vial and 96 ethanol to Buffer RA3 Concentrate and mix Reconstitute and store ly ophilized rDNase 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 No ethanol has been added after lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage e Reconstitute and store lyophilized rDNase according to in Poor RNA structions given in section 3 quality or yield Store other kit components at room temperature Storage at low temperatures may cause salt precipitation e Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influence A260 absorption as well as ratio A260 A 280 e For adsorption measurement use 5 mM Tris pH 8 5 as dilu ent Please see also Manchester KL 1995 Value of A A ratios for measure ment of purity of nucleic acids Biotechniques 19 208 209 Wilfinger WW Mackey K and Chomczyski P 1997 Effect of pH
9. 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this User Manual 5 2 Product description 6 2 1 The basic principle 2 2 Kit specifications 2 3 Handling preparation and storage of starting materials 11 2 4 Guideline for appropriate sample amount precipitation and resolubilization volume for protein isolation 13 2 5 Elution procedures 14 3 Storage conditions and preparation of working solutions 15 4 Safety instructions risk and safety phrases 17 5 Protocols 18 5 1 Total RNA and protein purification from cultured cells and tissue with NucleoSpin RNA Protein 18 5 2 Support protocol NucleoSpin RNA Protein Total RNA preparation from biological fluids e g serum culture medium 24 5 3 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 10 bacterial cells 25 5 4 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 5 x 107 yeast cells 27 5 5 Support protocol NucleoSpin RNA Protein Total RNA preparation from RNA ate treated samples 28 5 6 Support protocol NucleoSpin RNA Protein rDNase digestion in solution 29 6 Appendix 31 6 1 Protein quantification 31 6 2 Troubleshooting 39 6 3 References 43 6 4 Ordering information 44 6 5 Product use restriction warranty 45 MACHEREY NAGEL 10 2008 Rev 05 3 Total RNA and Protein Isolation 1 Components 1 1 Kit contents NucleoSpin RNA Protein Cat No Protein Precipitator PP Protein Solving Buffer PSB
10. 2 carboxyethyl phosphine and Dithiothreitol for use in protein biochemistry Analytical Biochemistry 273 73 80 The following publication describes a method for quantification of protein dis solved in sampel buffer such as PSB Karlsson JO Ostwald K Kabj rn C and Andersson M 1994 A method for protein assay in Laemmli buffer Analytical Biochemistry 219 144 146 MACHEREY NAGEL 10 2008 Rev 05 43 Total RNA and Protein Isolation 6 4 Ordering information Product Cat No Pack of NucleoSpin RNA Protein 740933 10 50 250 10 50 250 Protein Quantification Assay 740967 50 250 50 250 NucleoSpin RNA II 740955 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA DNA Buffer Set 740944 100 Buffer RP1 740934 50 50 ml Buffer RP1 740934 500 500 ml eee e Ay Buffer Set 740941 1 set rDNase Set 740963 1 set NucleoSpin Filters 740606 50 Collection Tubes 2 ml 740600 1000 Porablot See price list Blotting Paper See price list DISTRIBUTION AND USE OF THE NUCLEOSPIN RNA DNA BUFFER SET IN THE USA IS PROHIBITED FOR PATENT REASONS 44 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 6 5 Product use restriction warranty NucleoSpin RNA Protein kit components were developed designed distributed and sold FO
11. 5 prediluted 3 Assay Kit reducing sample corresponding 3 125 50 ug 0 625 10 pg pl Agent compatible to 5 ul original sample Method tested in MN laboratory for compatibility with PSB PSB TCEP and Laemmli buffer protein samples 32 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation Protein quantification assay Acceptable Input sample volume protein e g PSB TCEP amount per sample assay 100 ul of a 1 10 prediluted sample Protein concentration quantification range undiluted PSB TCEP sample corresponding to 10 ul 20 150 ug iginal Bio Rad DC Protein ee ae standard 4 standard assay or 2 15 jig ul Assay i 5 ul of a 1 10 prediluted 1 0 7 5 pg sample corresponding micro to 0 5 ul original sample micro testtube assay 100 pl of a 1 5 prediluted sample correspoding to 20 ul 20 150 ug Bio Rad RC DC original sample or standard 5 DEDEN standard assay 1 7 5 pg ul 25 ul of a 1 5 prediluted 5 0 37 5 yg sample corresponding micro to 5 ul original sample micro testtube assay g ere Erotad lan 20 ul 5 35 0 25 1 75 g l Assay Kit H Hg f3 HO H G Bioscience 7 SPN Protein 1 10 pl 0 5 10 ug 0 05 10 ug pl Assay nio Rad r roren 100 pl of 1 20 prediluted 8a Assay Standard S le derd ondin 20 140 4 28 yg ul Assay Proceaure to Y l oti inal a i P9 n Bradford Pe P Bio Rad Protein 8b Assay Mi
12. M sodium acetate pH 5 2 and 2 5 volumes of 96 100 ethanol to one volume of sample Mix thoroughly Incubate several minutes to several hours at 20 C or 4 C Note Choose long incubation times if the sample contains low RNA concentration Short incubation times are sufficient if the sample contains high RNA concentration Centrifuge for 10 min at max speed Wash RNA pellet with 70 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 30 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 6 Appendix 6 1 Protein quantification Quantification of protein dissolved in sample buffer such as PSB PSB TCEP or tra ditional Laemmli buffer is occasionally required prior to SDS PAGE and Western Blot analysis However major protein quantification assays are influenced by or are incom patible with SDS and or reducing agents commonly present in protein sample buffers used for SDS PAGE A protein quantification procedure has to be chosen carefully to ensure appropriate compatibility of the method with the protein dissolution solution The NucleoSpin RNA Protein procedure allows several protein quantification methods at different steps of the procedure e Quantification of protein dissolved in PSB PSB TCEP or Laemmli buffer recommended e Quantification of protein dissolved in alternative protein dissolution buffers 1 SDS or 8 M urea e Quantification of protein within the column flow through
13. 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 down stream 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 Western Blot analysis and quantification with recommended methods 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 chao tropic ions This lysis buffer immediately inactivates virtually all enzymes e g RNases and proteases which are present in almost all biological materials The buffer dis solves 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 harm ful 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 an rDNase solution which is directly applied
14. any precipitate Bind RNA For each preparation take one NucleoSpin RNA Protein Column light blue ring placed in a Collection Tube and load the lysate Centrifuge for 30 s at 11 000 x g Place the NucleoSpin RNA Protein Column in a new Collection Tube 2 ml RNA and DNA are bond to the column membrane protein 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 For RNA isolation continue with step 6 It is recommended to continue the RNA isolation protocol first and to perform the 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 350 ul 70 EtOH Mix Load sample 30s 11 000 x g MACHEREY NAGEL 10 2008 Rev 05 19 NucleoSpin RNA Protein Further steps for RNA purification steps 6 9 6 Desalt silica membrane Add 350 pl MDB Membrane Desalting Buffer and cen trifuge at 11 000 x g for 1 min to dry the membrane Salt removal will enhance the effectivity of the following rDN ase digest much more effective If the column outlet has come into contact with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g Digest DNA Prepare rDNase reaction mixture in a sterile micro centrifuge
15. i e prior to protein pre cipitation at step 5 of the standard protocol For most reliable results and convenience we recommend the MACHEREY NAGEL Protein Quantification Assay for ordering information see section 6 4 to quantify protein dissolved in PSB PSB TCEP or Laemmli buffer Quantification of protein dissolved in PSB or PSB TCEP The concentation of protein dissolved in PSB PSB TCEP or Laemmli buffer can be determied with several methods Below you find a choice of quantification methods which are compatible with PSB PSB TCEP and Laemmli buffer but not all of the methods show the same sensitivity The following list compares the different sensitivies and gives assistance to find out the most suitable protein quantification assay 1 Protein Quantification Assay highly recommended method This is the most sensitive and convenient method for protein quantification in PSB or PSB TCEB Highly recommended due to sensitivity and high compatibility For ordering information see section 6 4 2 Method adapted from the publication Karlsson et al 1994 See below for details 3 Pierce BCA Protein Assay Kit reducing agent compatible Dilute the protein sample 1 5 with water to enable compatibility MACHEREY NAGEL 10 2008 Rev 05 31 Total RNA and Protein Isolation 4 Bio Rad DC Protein Assay Dilute the protein sample 1 10 with water to enable compatibility 5 Bio Rad RC DC Protein Assay Dilute the protein s
16. is sufficient for subsequent determination of two cali bration curves MACHEREY NAGEL 10 2008 Rev 05 37 Total RNA and Protein Isolation Add 20 ul of each dilution series sample 1 8 in microtiter plate wells 2 Add 20 ul of samples protein dissolved in PSB TCEP with unknown protein concentration to further wells alternatively 1 60 ul 3 Add 40 ul PSB TCEP to each well Final volume 60 ul alternatively add 0 59 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 extinction at 570 nm N 9 9 A Determine protein concentration of samples in relation to dilution series a ee 5 E t E m 0 01 F fa F Fa r ii 0 001 L e 1 titi 0 1 1 10 100 BSA amount per well yg Figure 1 BSA standard curve for determination of protein in Protein Solving Buffer PSB Measurement of extinction in the range of 530 700 nm is suitable and will typically result in correlation coef ficients of 0 99 concentration of BSA dilution series vs obtained absorption values 38 MACHEREY NAGEL 10 2008 Rev 05 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 degraded
17. not supplied Protein Solving Buffer with reducing agent PSB TCEP e BSA Bovine Serum Albumin not supplied e Multititer plate not supplied MACHEREY NAGEL 10 2008 Rev 05 35 Total RNA and Protein Isolation Composition of PSB TCEP e 125 mM BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane e 2 SDS sodium dodecyl sulphate 50 mM TCEP Tris 2 carboxyethyl phosphine Hydrochloride e 20 glycerol e 0 01 brome phenol blue e pH6 8 Note The composition of the previously used Protein Loading Buffer PLB has been improved and is now called Protein Solving Buffer PSB reduced concentration of SDS and bromphenol blue The change in composition has increased the compatibility with protein quantification methods see above For details on the composition of previous Protein Loading Buffer PLB contact our technical service The volume of PSB TCEP included in the respective kit sizes is sufficient for the following procedures Required volume of PSB TCEP Cat No 740933 10 740933 50 740933 250 Step Solubilization of protein pellet in 100 pl 1000 ul 5 ml 25 ml see section 5 1 step 10 x 100 ul 50 x 100 ul 250 x 100 ul 13 BSA dilution series sufficient for determi 448 ul 0 45 ml 5 x 0 45 ml nation of two calibra 1x97 5 ul 1x97 5 ul 1x97 5 ul tion curves see 6x50 ul 6x50 ul 6x50 ul page 37 Fill up of all protein ae a an Reece emi om sample is quantified
18. onto the silica membrane during the preparation RNase free rDNase 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 in denatured form with a special buffer Protein Precipitator PP which effectively precipitates pro tein After a washing step the protein pellet is dissolved in Protein Solving Buffer PSB containing the odourless reducing agent TCEP The protein can thus readily be ap plied to SDS PAGE analysis The kit is not recommended for isolation of native proteins The RNA and protein preparation using NucleoSpin RNA Protein kits can be per formed 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 gen eral lab ware fingerprints and dust To ensure RNA stability keep RNA frozen at 20 C 6 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation for short term or 70 C for long term storage Recovered Protein dissolved in Protein Solving 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 for RNA
19. Add 7 5 ml PSB each 16 MACHEREY NAGEL 10 2008 Rev 05 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 rDNase rDNase x Xi May cause sensiti R 42 43 S 22 24 lyophilized zation by inhalation and skin contact Buffer RP1 Guanidine x Xn Harmful by inhala R 13 thiocyanate tion in contact with 20 21 22 the skin and if swallowed Buffer RA2 Guanidine x Xn Flammable R 10 S 7 13 16 thiocyanate Harmful by inhala 20 21 22 ethanol lt 25 tion in contact with the skin and if swallowed MDB Guanidine thio Flammable R10 S 7 16 cyanate lt 10 ethanol lt 10 Reducing Tris 2 carboxy x Xi Causes burns R 34 S 26 27 Agent TCEP ethyl phosphine 36 37 39 hydrochloride Risk phrases R10 Flammable R 34 Causes burns Harmful by Ipalauon ip May cause sensitization by inha R 20 21 22 contact with the skin and if R 42 43 ay N y lation and skin contact swallowed Safety phrases S7 Keep container tightly closed S 24 Avoid contact with the skin Keep away from food drink S43 and animal feedstuffs S16 Keep away from sources of ignition No Smoking S 22 Do not breathe dust Hazard labeling not neccessary if quantity p
20. Balen D Brzica H Krick W Burckhardt BC Sabolic and Burckhardt G 2008 Identification of a new urate and high affinity nicotinate trans porter human organic anion transporter 10 hOAT10 SLC22A13 J Biol Chem published 14 April 2008 10 1074 jbc M800737200 Weiske J Albring KF and Huber O 2007 The tumor suppressor Fhit acts as a re pressor of _ catenin transcriptional activity PNAS Dec 2007 104 20344 20349 The following publications show the general usefulness of the parallel extraction of DNA RNA and protein from small and precious samples 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 188 338 343 Banerjee S Smallwood A Chambers AE and Nicolaides K 2003 Quantitative re covery of immunoreactive proteins from clinical samples following RNA and DNA isola tion BioTechniques 35 3 450 456 Hoemann CD Sun J Chrzanowski V and Buschmann MD 2002 A multivalent as say to detect glycosaminoglycan protein collagen RNA and DNA content in milligam samples of cartilage or hydrogel based repair cartilage Analytical Biochemistry 300 1 10 The following publication decribes the Reducing Agent TCEP Getz EB Xiao M Chakrabarty T Cooke R and Selvin PR 1999 A comparison be tween the sulfhydryl reductants Tris
21. CEP see section 6 1 for composition This provides sufficient molar excess to reduce peptide and protein disulfide bonds effec tively within a few minutes in a range up to a protein concentration of approximately 1 pg pl 2 5 Elution procedures It is possible to adapt elution method and elution volume of water to 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 on ice and always kept on ice for optimal sta bility 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 14 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 3 Storage conditions and preparation of working solutions Attention Buffers RP1 RA2 and MDW contain guanidine thiocyanate Wear gloves and goggles Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year Store Reducing Agent TCEP at 4 C on arrival All other kit components should be stored at
22. P PSB TCEP is stable for several days at room temperature 18 25 C and several months at 4 C For long term storage of PSB TCEP keep at 20 C For 50 and 250 prep kits For better handling PSB TCEP may be transferred into the original PSB vial with screw Cap MACHEREY NAGEL 10 2008 Rev 05 15 Total RNA and Protein Isolation e If SDS PAGE under non reducing conditions is intended consider the follow ing A Omit addition the Reducing Agent TCEP to Buffer PSB B Omit addition of B mercaptoethanol to Lysis Buffer RP1 e f other reducing agents than TCEP are preferred e g DTT 8 mercaptoetha nol appropriate amounts should be added to PSB Please consider limited stability of DTT compared to TCEP e f PSB TCEP is turbid warm up PSB TCEP to gt 25 C before use until solu tion is completely clear i e all precipitate is dissolved completely PSB TCEP has a half life of approximately 5 months if stored at 4 C and approximately 7 months if stored at 20 C NucleoSpin RNA Protein 10 preps 50 preps 250 preps Cat No 740933 10 740933 50 740933 250 Wash 3x 25 ml 5 ml 12 5 ml Bufer RAS Add 20 ml ethanol Add 50 ml ethanc 99 100 ml ethanol Concentrate to each vial rDNase 1 vial size C 1 vial size D er i RNase free Add 230 ul Add 540 pl i roe lyphilized RNase free H O RNase free H O Rit 2 to each vial Reducing 2x14 mg 107 mg 2 x 107 mg Agent TCEP Add 1 ml PSB each Add 7 5 ml PSB
23. R RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organ ism or for clinical use diagnostic prognostic therapeutic or blood banking Itis rather the responsibility of the user to verify the use of the NucleoSpin RNA Protein kit 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 specifica tions 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 im proper 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 AN
24. Y 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 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 spe cial 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 state ments 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 10 2008 Rev 05 45 Total RNA and P
25. ample 1 5 with water to enable compatibility 6 Serva ProtaQuant Assay Kit According to manufacturer s instructions this assay should be compatible with PSB PSB TCEP and Laemmli buffer samples 7 G Biosciences SPN Protein Assay According to manufacturer s instructions this assay should be compatible with PSB PSB TCEP and Laemmli buffer samples 8 Bio Rad Protein Assay Bradford This method has a very low tolerance towards SDS 0 1 SDS for the Standard Assay Procedure Therefore PSB PSB TCEP Laemmli buffer samples have to be diluted considerably with water to reduce interference After dilution of the sample 1 20 with water protein can be quantified with the standard assay procedure The microassay procedure however is not compatible with such samples even after 1 50 dilution of the sample with water Compatibility of protein quantification methods with PSB and PSB TCEP samples Acceptable Protein concentration Protein Input sample volume BEN protein quantification range quantification e g PSB TCEP amount per undiluted PSB TCEP assay sample assay sample Protein 0 03 1 g l 1 Quantification 20 pl standard 0 6 20 ug standard Assay highly 1 60 ul optional 0 01 20 ug l recommended optional 0 03 1 pg pl 2 Karlsson protocol 20 ul standard 0 6 20 pg standard recommended 1 60 ul optional 0 01 20 pg ul optional Pierce BCA Protein 25 ul of a 1
26. and DNA isolation in conjunction with NucleoSpin RNA II NucleoSpin RNA XS NucleoSpin RNA Plant or NucleoSpin RNA Protein This patented 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 of the NucleoSpin RNA DNA Buffer Set 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 A A ratio generally exceeding 1 9 measured in TE buffer pH 7 5 The isolated RNA is ready to use for applications like reverse transcriptase PCR RT PCR primer extension or RNase protection assays e The isolated protein is ready to use for SDS PAGE Western Blot analysis and protein quantification with the Protein Quantification Assay see ordering infor mation DISTRIBUTION AND USE OF THE NUCLEOSPIN RNA DNA BUFFER SET IN THE USA IS PROHIBITED FOR PATENT REASONS PCR is patented by Roche Diagnostics MACHEREY NAGEL 10 2008 Rev 05 7 Total RNA and Protein Isolation Table 1 Kit specifications at a glance Parameter NucleoSpin RNA Protein Up to 5 x 10 cells Sample material Up t
27. and ionic strength on the spectrophotometric assess ment of nucleic acid purity Biotechniques 22 474 481 MACHEREY NAGEL 10 2008 Rev 05 39 Total RNA and Protein Isolation Poor RNA quality or yield continued Sample material Sample material was not stored properly Whenever possible use fresh material If this is not possible flash freeze the sam ples in liquid N Samples should always be kept at 70 C Never allow tissues to thaw before addition of Buffer RP1 Perform disruption of samples in liquid N Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Clogged NucleoSpin Column Poor RNA quality or yield Sample material Too much starting material used Overloading may lead to de creased overall yield Reduce amount of sample material or use larger volume of Buffer RP1 Insufficient disruption and or homogenization of starting mate rial Ensure thorough sample disruption and use NucleoSpin Filters Filters L for easy homogenization of disrupted starting material Contamination rDNase not active Reconstitute and store lyophilized rDNase according to in structions given in section 3 rDNase solution not properly applied of RNA with Pi DN luti ioci h f the sili genomic DNA ipette rDNase solution directly onto the center of the silica mem
28. ate sample amount precipitation and resolubilization volume for protein isolation The following table 5 shall serve as a first guide for choosing appropriate amounts of sample material precipitation volume and resolubilization volume Depending on sample type and downstream application e g Coomassie or silver stain sensitivity of antibody detection system appropriate volumes might deviate from the table below and have to be determined experimentally Table 5 Guideline for appropriate sample amount Cultivated cells Animal tissue Plant tissue Amount of e g garden e g HeLa e g liver cress leaf Sample 10 105 104 30mg 3 mg 0 3 mg 100 mg 10 mg 1mg Lysis Buffer RP1 incl reducing 350 jil agent Ethanol 350 ul Column flow through to be 35 pl 350 pl 700 ul 35 pl 350 ul 700 pl 35 pl 350 pl 700 ul precipitated Volume of Protein Precipitator PP Buffer PSB used for protein pellet 100 pl 100 pl 20 pl 100 ui 100 ul 20 pl 100 pi 100 pi 20 ul solubilization 35 pl 350 pl 700 jl 35 ul 350 pl 700 pl 35 pl 350 pl 700 pl Protein sample to be analysed on SDS PAGE with 10 Coomassie stain Protein sample to be analyzed on 4 ul SDS PAGE with Hi silver stain Protein sample analyzed on 1 10 jil Western Blot Protein pellets with a diameter of up to approximately 1 2 mm in size are i
29. brane Too much cell material used e Reduce quantity of cells or tissue used 40 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation DNA detection system too sensitive Contamination of RNA with genomic DNA continued The amount of DNA contamination is significantly reduced during the on column rDNase 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 sys tem 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 1 kb fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the rDNase is applied however a strong PCR fragment is obtained if rDNase 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 500 bp or intron spanning primers if possible Use support protocol 5 6 for subsequent rDNase diges tion in solution Carryover of ethanol or salt Suboptimal performance of RNA in downstream Do not let the flow through touch the column outlet after the second RA3 wash Be sure to centrifuge at the c
30. cr assay Not recommended E E Procedure Bradford Method tested in MN laboratory for compatibility with PSB PSB TCEP and Laemmli buffer protein samples Method compatible with PLB TCEP protein samples refering to manufacturer s product information Not tested in MN laboratories MACHEREY NAGEL 10 2008 Rev 05 33 Total RNA and Protein Isolation Quantification of protein within the column flow through Alternative to quantification of protein dissolved in PSB TCEP protein can be quantified within the ethanolic lysate column flow through Knowlege of protein concentration in the column flow through helps to choose an appropriate volume for subsequent pre cipitation with Protein Precipatator PP The following methods are suitable to quantify protein in the column flow through 1 Bio Rad Protein Assay Bradford The standard assay procedure is compatibel with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 The microassay procedure is not compatible the column flow through 2 Pierce BCA Protein Assy Kit reducing agent compatible This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 3 Bio Rad DC Protein Assay This method is compatible with the colu
31. deally suited for subsequent solubilization Protein pellets exceeding volumes of approximately 10 ul should be avoided as large protein pellets are harder to dissolve than small pellets To obtain small protein pellets adapt the volume of column flow through in respect to the amount of sample material Commonly small and even invisible protein pellets yield sufficient protein for SDS PAGE and Western Blot analysis MACHEREY NAGEL 10 2008 Rev 05 13 Total RNA and Protein Isolation Solubilization of protein pellets and reduction of protein disulfide bonds The NucleoSpin RNA Protein kit provides a protein sample buffer Protein Solving Buffer PSB and the Reducing Agent TCEP The Protein Solving Buffer PSB is similar in composition and function to the buffer com monly known as Laemmli buffer For most applications PSB may be substituted by Laemmli buffer However for applications with large protein pellets gt approx 1 mm PSB is recommended TCEP is a powerful multi purpose and odourless reducing agent It is non volatile and unlike commonly used reducing agents like DTT and mercaptoehanol resistant to air oxidation TCEP reduces disulfide bonds as effectively as dithiothreitol DTT TCEP reduces even most stable water soluble alkyl disulfides selectively and completely over a wide pH range Solubilization of TCEP in PSB according to the instruction results in a PSB TCEP solution with a concentration of 50 mM T
32. er For further information see Material Safety Data Sheet In case of contact with eyes S 26 rinse immediately with plenty of water and seek medical advice S 27 S 36 37 39 Take off immediately all contami nated clothing Wear suitable protective clothing gloves and eye face protection bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 MACHEREY NAGEL 10 2008 Rev 05 17 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 Before starting the preparation e Check if Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 1 Homogenization of sample Disrupt up to 30 mg of human animal tissue or up to 100 mg of plant tissue for homogenization methods Disrupt see section 2 3 sample Up to 5 x 10 eukaryotic cultured cells are collected by centrifugation and lysed by addition of Buffer RP1 direct ly 2 Cell lysis Add 350 ul Buffer RP1 and 3 5 pl B mercaptoethanol B ME to the cell pellet or to ground tissue and vortex 350 pI RP1 vigorously 3 5 ul B ME Note As alternative to B ME the reducing agent DTT or TCEP may be use instead of B ME Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RP1
33. er for ordering information see section 6 4 The procedure presented below based on the publication of Karlsson et al 1994 is also suitable for quantification of protein dissolved in Protein Solving Buffer PSB TCEP and may be followed alternatively Nucleic acids disturb protein quantification as described by Karlsson et al 1994 Protein samples obtained 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 concentra tion in the range 5 ng pl 20 j g ul by using variable sample volumes of 1 60 ul Recommended sample volume For protein concentra Protein amount protein dissolved in PLB TCEP tion in the range of per well 60 ul 0 01 0 33 pg pl 0 6 20 ug 20 ul 0 03 1 00 pg pl 0 6 20 ug 1 ul 0 6 20 ug pl 0 6 20 ug For the primary determination of protein concentration of the sample measure different amounts e g 2 ul 10 pl and 50 ul This assures that at least one value of the three amounts tested falls within the range of the calibration curve Further for a first rough estimation of expected protein yield also consider table 4 in section 2 2 an table 5 in section 2 4 Material e TCA 60 Trichloracetic acid
34. g of undisrupted animal tissue should exclusively be done in 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 preparation has started The spinning rotor disrupts and simultaneously homogenizes MACHEREY NAGEL 10 2008 Rev 05 11 Total RNA and Protein Isolation the sample by mechanical shearing 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 5 By this treatment the robust cell walls of these organ isms 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 treat ment with lytic enzymes or the cultivation conditions After lysis homogenization is achieved by the use of a NucleoSpin Filter or the syringe needle method 12 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 2 4 Guideline for appropri
35. le RNase free tips Equipment e Manual pipettors e Centrifuge for microcentrifuge tubes e Vortex mixer e Thermal heating block e Equipment for sample disruption and homogenization e Personal protection equipment lab coat gloves goggles Additional material is furthermore needed for protein quantification see section 6 1 1 3 About this User Manual It is strongly recommended reading the detailed protocol sections of this User Manual if the NucleoSpin RNA Protein kit is used for the first time Experienced users how ever may refer to the Protocol at a glance instead The Protocol at a glance is de signed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 10 2008 Rev 05 5 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 quantifica tion of RNA and protein are often hampered by the small sample size and the ne cessity 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 and RNA are isolated without splitting the sample prior to protein
36. les 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 protein yield Centrifuge for 5 min at 11 000 x g 11 Wash protein 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 precipitate might appear as no visible pellet e g for 10 000 cells 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 See also section 2 4 how to avoid very large protein pellets 12 Dry protein pellet Dry precipitate for 5 10 min at room temperature keep lid open Note Large pellet
37. mn flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 4 Bio Rad RC DC Protein Assay This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 5 Roti Nanoquant Assay This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 Protein quantification in alternative protein pellet dissolution buffers The use of the PSB or PSB TCEP buffer is not mandatory for solving proteins Precipitated protein protein pellets may be dissovled in alternative solutions such as 1 SDS or 8 M urea or in urea thiourea CHAPS buffers as used for 2 D electro phoresis However depending on the target protein the yield of solubilized protein may be reduced compared to PSB or PSB TCEP Check manufacturers product information to ensure compatibility of your protein quantifiction assay with your alternative protein dissolving solution 34 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation Protocol for protein quantification For most reliable results and convenience we recommend the MACHEREY NAGEL Protein Quantification Assay to quantify protein dissolved in PSB PSB TCEP or Laemmli buff
38. mple for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contami nating genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections the target gene is of a very low expression level e the amplicon is relatively small lt 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However strin gent RNase control and subsequent repurification of the RNA in order to remove buf fer salts DNase and digested DNA are usually required The high quality recombinant RNase free DNase rDNAse in the NucleoSpin RNA kits facilitates such a digestion in solution in order to remove even traces of contami nating DNA A Digest DNA reaction setup Add 6 pl Reaction Buffer for rDNase and 0 6 ul rDNase to 60 pI eluted RNA Alternatively premix 100 ul Reaction Buffer for rDNase and 10 ul rDNase and add 1 10 volume to one volume of RNA eluate B Incubation Incubate for 10 min at 37 C MACHEREY NAGEL 10 2008 Rev 05 29 NucleoSpin RNA Protein Repurification of RNA Repurify RNA with a suitable RNA clean up procedure e g by use of the NucleoSpin RNA Clean up or NucleoSpin RNA Clean up XS kit see ordering information or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3
39. nd centrifuge at 11 000 x g for 1 min If higher RNA concentrations are desired elution can be done with 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 13 250 ul RA3 2 min 11 000 x g 60 pl RNase free H O 1 min 11 000 x g Perform steps 1 5 of the NucleoSpin RNA Protein kit standard protocol homogenization cell lysis lysate filtration adjusting of nucleic acid bind ing condition and binding of nucleic acids to the NucleoSpin RNA Protein Column Use the NucleoSpin RNA Protein Column flow through of step 5 i e the etha nolic lysate which has been passed throught the RNA binding column and is as such deprived of nucleic acids as starting point for protein precipitation step 10 MACHEREY NAGEL 10 2008 Rev 05 21 NucleoSpin RNA Protein 10 Precipitate protein Transfer an appropriate amount 10 700 ul of flow though into a fresh Collection Tube 1 5 ml supplied See section 2 4 as guideline for choosing an appropriate amount Add one volume PP Protein Precipitator Mix vigorously Incubate mixture at room temperature for approximately 10 min 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 samp
40. nd or reducing agents pres ent in Protein Loading Buffer e Use a suitable quantification method as described in section 6 1 e f an other protein dissolution buffer than PSB or PSB TCEP has used for dissolving the protein pellet ensure compatibility of your buffer and quantification method of choice No protein precipitate pellet visible A small sample amount was used and or a small volume of column flow through was used for precipitation e Formation of a visible protein pellet is not required for suf ficient protein recovery Even invisible protein pellets com monly yield enough protein for SDS PAGE and Western Blot analysis PSB TCEP PSB TCEP may form a precipitate at temperatures below 18 C turbid or par e Warm up 25 C to dissolve turbidity completely tially solidified Protein was resolubilized in water No low e Due to the strongly denatured form of the protein the solubil protein yield ity in water is significantly reduced Use PSB TCEP for pro tein solubilization 42 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation 6 3 References The following publications cite the use of the NucleoSpin RNA Protein kit Rodriguez Jim nez FJ Moreno Manzano V Lucas Dominguez R and S nchez Puelles JM 2008 Hypoxia Causes Down Regulation of Mismatch Repair System and Genomic Instability in Stem Cells Stem Cells May 2008 10 1634 stemcells 2007 1016 Bahn A Hagos Y Reuter S
41. o 30 mg human animal tissue Up to 100 mg plant tissue RNA yield Up to 70 ug Protein yield Up to 1200 ug Elution volume RNA 40 100 ul Resolubilization volume protein 10 100 ul Binding capacity RNA 200 ug Preparation time RNA lt 30 min 6 preps Preparation time protein 35 min 6 preps Format Mini spin column e The standard protocol section 5 1 allows purification of up to 70 ug of to tal RNA per NucleoSpin RNA Protein Column from up to 5 x 10 cultured cells 30 mg of human animal tissue or 100 mg of plant tissue 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 Intron spanning primers for RT PCR are preferable if possible e RNA prepared with NucleoSpin RNA Protein is generally free of residual DNA However minute traces of DNA may remain if large amounts of material rich in nucleic acids are used If the isolated RNA will be used as template in a RT PCR reaction we recommend using lower quantities of sample material depending on cell or tissue type in the range of 1 x 10 cells or 10 mg of tissue resulting in about 20 ug of RNA 8 MACHEREY NAGEL 10 2008 Rev 05 Total RNA and Protein Isolation e The kit can be used for preparing RNA from different amounts of sample mate rial according to table 2 Table 2 Use of different amounts of sample material
42. or bitol 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 concentra tion depending on the yeast strain 2 Cell lysis Add 350 pl Buffer RP1 and 3 5 pl B mercaptoethanol ME to the suspen sion and vortex vigorously As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RP1 3 Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters Place NucleoSpin Filters in Collection Tubes 2 ml 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 microcentrifuge tube not included Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe Due to the much higher concentration of genome equivalents in a nucleic acid prepa ration of yeasts compared with cultured cells or tissue material it may be necessary to use a lower quantity of cells for the preparation Proceed with step 4 of the NucleoSpin RNA Protein standard protocol sec tion 5 1 MACHEREY NAGEL 10 2008 Rev 05 27 NucleoSpin RNA Protein
43. orresponding speed for the respective time in order to remove ethanolic Buffer RA3 completely Check if Buffer RA3 has been equilibrated to room tempera ture before use Washing at lower temperatures lowers ef ficiency of salt removal by Buffer RA3 experiments Store isolated RNA properly 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 MACHEREY NAGEL 10 2008 Rev 05 41 Total RNA and Protein Isolation Trouble with resolubilization of precipi tated protein in PSB TCEP Protein pellets exceeding several millimeters in size are hard to dissolve Use smaller volumes of column flow through for protein pre cipitation in order to obtain small sized pellets Even invisible protein pellets commonly yield enough protein for SDS PAGE and Western Blot analysis Protein dis solved in PSB TCEP escapes SDS PAGE gel slot im mediately after loading Protein pellet has not been dried sufficiently and contains residual ethanol e Increase drying time or decrease pellet size by precipitating a smaller volume of column flow through Unclear results with commonly used protein quantification systems Most commonly used protein quantification systems are incom patible with concentrations of SDS a
44. r TCEP within the Lysis Buffer RP1 3 Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters violet rings Place NucleoSpin Filters in Collection Tubes apply mix ture 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 5 times through a 0 9 mm needle 20 gauge fitted to a syringe MACHEREY NAGEL 10 2008 Rev 05 25 NucleoSpin RNA Protein Adjust RNA binding conditions Add 350 pl ethanol 70 and mix by pipetting up and down approx 5 times 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 Proceed with step 5 of the NucleoSpin RNA Protein standard protocol sec tion 5 1 26 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein 5 4 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 5 x 10 yeast cells Before starting the preparation Check if Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 1 Homogenization of sample Harvest 2 5 ml YPD culture 5 000 x g 10 min Resuspend pellet in s
45. room temperature 20 25 C and are stable up to one year Storage at lower temperatures may cause precipita tion of salts Check that 70 ethanol is available as additional solution to adjust binding conditions in the RP 1 lysate Check that 50 ethanol is available as additional solution to wash the protein pellet Before starting any NucleoSpin RNA Protein protocol prepare the following rDNase RNase free Add indicated volume of RNase free H O see table be low to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the rDNase Be careful not to mix rDNase vig orously as rDNase 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 Wash Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to Buffer RA3 Concentrate Mark the label of the bottle to indicate that the ethanol is added Store Wash Buffer RA3 at room temperature 20 25 C for up to one year Protein Solving Buffer PSB and Reducing Agent TCEP For SDS PAGE un der reducing conditions most common type of SDS PAGE transfer PSB from one vial to one vial of the Reducing Agent TCEP Mix gently to avoid excessive foaming until the reducing agent is dissolved completely this process will re quire several minutes Protein Solving Buffer containing Reducing Agent TCE
46. rotein 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 your 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 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 Trademarks NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG RNA ater is a registered trademark of AMBION Inc SPN is a registerede trademark of G Biosciences All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 46 MACHEREY NAGEL 10 2008 Rev 05
47. s e g complete precipitation of 700 ul column flow through form a 30 mg liver sample need longer drying duration Samples which are dried incom plete may cause problems when loading the sample onto the gel due to residual ethanol content No problems with over drying have been observed with small sized pellets See also section 2 4 how to avoid very large protein pellets 22 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein 13 Prepare protein sample Add 20 100 pl PSB TCEP Protein Solving Buffer containing reducing agent Assure that PSB TCEP is clear not turbid If necessary warm PSB TCEP to gt 25 C to dissolve turbidity See section 2 4 as guideline for choosing an appropriate amount of PSB TCEP for dissolving of protein pellets 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 denatur ation Let sample cool down to room temperature 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 residual precipitates at this stage Protein will be in the supernatant Do not centri fuge samples at temperatures lt 18 C SDS may precipitate at
48. se and Reducing Agent TCEP were prepared according to section 3 1 Homogenization of sample Not necessary 2 Cell lysis Add 350 ul Buffer RP1 to 100 pl of sample and vortex vigorously 3 Filtration of the lysate Not necessary 4 Adjust RNA binding conditions Add 350 pl ethanol 70 to the lysate and mix by pipetting up and down ap prox 5 times Proceed with step 5 of the NucleoSpin RNA Protein standard protocol sec tion 5 1 24 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein 5 3 Support protocol NucleoSpin RNA Protein Total RNA preparation from up to 10 bacterial cells Before starting the preparation Check if Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 1 Homogenization of sample Resuspend the bacterial cell pellet Gram negative strains in 100 pl TE buffer 10 mM Tris HCI 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 Depending on the bacterial strain it may be neces sary to optimize incubation time and lysozyme concentration 2 Cell lysis Add 350 pl Buffer RP1 and 3 5 pl B mercaptoethanol ME to the suspen sion and vortex vigorously As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT o
49. this temperature Recover supernatant for further analysis See section 6 1 for suitable protein quantification methods Note At this stage samples can be stored at 20 C for several months or at 4 C for several 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 Due to the strong denaturing purification method protein is precipitated in denatured form with reduced solubility in water Therefore resolubilization of the protein pellet in PSB TCEP or in traditional Laemmli buffer is recommended The use of Protein Solving Buffer PSB is not mandatory for dissolving protein Alternatively to PSB PSB TCEP or Laemmli buffer precipitated protein can be dissolved in 1 SDS or 8 M urea Further the protein pellet can be dissolved in urea thiourea CHAPS buffers as used for 2 D electrophoresis However depending on the target protein the overal yield of solubilized protein may be reduced compared to PSB or PSB TCEP used as dissolving agent MACHEREY NAGEL 10 2008 Rev 05 23 NucleoSpin RNA Protein 5 2 Support protocol NucleoSpin RNA Protein Total RNA preparation from biological fluids e g serum culture medium Before starting the preparation Check if Wash Buffer RA3 rDNa
50. tube not provided For each isolation add 10 pl reconstituted rDNase also see section 3 to 90 ul Reaction Buffer for rDNase Mix by flicking the tube Apply 95 pl rDNase 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 Add 200 pl Buffer RA2 to the NucleoSpin RNA Protein Column Centrifuge for 30s at 11 000 x g Place the NucleoSpin RNA Protein Column into a new Collection Tube 2 ml Buffer RA2 will inactivate the rDNase Add 600 pl Buffer RA3 to the NucleoSpin RNA Protein Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the NucleoSpin RNA Protein Column back into the Collection Tube 350 pl MDB 95 ul rDNase reaction mixture RT 15 min 200 pl RA2 30s 11 000 x g c H gt 600 pl RA3 30 s 11 000 x g 20 MACHEREY NAGEL 10 2008 Rev 05 NucleoSpin RNA Protein Add 250 pl Buffer RA3 to the NucleoSpin RNA Protein Column Centrifuge for 2 min at 11 000 x g to dry the membranecompletely Placethe NucleoSpin RNA Protein Column into an RNase free Collection Tube 1 5 ml supplied If for any reason the liquid level in the Collectio Tube has reached the NucleoSpin RNA Protein Column after centrifu gation discard flow through and centrifuge again 9 Elute highly pure RNA Elute the RNA in 60 pl RNase free H O supplied a
51. ture medium in order to allow full lysis activity of the lysis buffer To trypsinize adherent growing cells Aspirate cell culture medium and add an equal amount of PBS in order to wash the cells Aspirate PBS Add 0 1 0 3 trypsin in PBS and incubate for an 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 Human animal and plant 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 re duced 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 presence of liquid N Take care that the sample does not thaw during or after grinding or weighing and add the fro zen powder to an appropriate aliquot of Buffer RP1 containing B mercaptoethanol and mix immediately The broken up tissue must then be homogenized with a NucleoSpin Filter Filter L or by passing 5 through a 0 9 mm syringe needle Thawin
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