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CST Protocols & Troubleshooting Guides

1. 4 Vortex briefly and let stand for 15 min at room temperature 5 Add 1 ml of 0 1 Triton X 100 to each tube 6 Vortex and let stand for 30 min at room temperature 7 Add 1 ml incubation buffer 8 Pellet cells by centrifugation and aspirate supernatant 9 Repeat steps 7 and 8 10 Resuspend cells in ice cold 50 methanol in PBS store methanol solution at 20 C until use 11 Incubate at least 10 min on ice subpopulations Cytometry A 67 4 17 12 Proceed with staining or store cells at 20 C in 50 methanol 08 2013 Cell Signaling Technology Inc Triton X 100 is a registered trademarke of the Dow Chemical Company www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan IMPORTANT Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use on cultured cell lines IF IC paraffin embedded samples IF P or frozen tissue sections IF F Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution This protocol is recommended for both unconjugated and fluorophore conjugated antibodies NOTE Some CST antibodies work optimally using an alternate protocol Please see product datasheet for product specific recommendations A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RO
2. DAB Substrate Kit 8059 B Deparaffinization Rehydration NOTE Do not allow slides to dry at any time during this procedure 1 Deparaffinize hydrate sections a Incubate sections in three washes of xylene for 5 min each b Incubate sections in two washes of 100 ethanol for 10 min each c Incubate sections in two washes of 95 ethanol for 10 min each 2 Wash sections two times in dH 0 for 5 min each 08 2013 Cell Signaling Technology Inc SignalStain and CST are trademarks of Cell Signaling Technology Tween 20 is a registered trademark of ICI Americas Inc C Antigen Unmasking NOTE Consult product datasheet for specific recommendation for the unmasking solution protocol 1 For Citrate Bring slides to a boil in 10 mM sodium citrate buffer pH 6 0 maintain at a sub boiling temperature for 10 min Cool slides on bench top for 30 min 2 For EDTA Bring slides to a boil in 1 mM EDTA pH 8 0 follow with 15 min at a sub boiling temperature No cooling is necessary 3 For TE Bring slides to a boil in 10 mM Tris 1 mM EDTA pH 9 0 then maintain at a sub boiling temperature for 18 min Cool at room temperature for 30 min 4 For Pepsin Digest for 10 min at 37 C D Staining NOTE Consult product datasheet for recommended antibody diluent 1 Wash sections in dH three times for 5 min each 2 Incubate sections in 3 hydrogen peroxide for 10 min 3 Wash sections in dH 0 two times for 5 min each 4 Wash section
3. 10X Tris Glycine Transfer Buffer 12539 To prepare 1 L 1X Transfer Buffer add 100 ml 10X Transfer Buffer 200 ml methanol 700 ml dH 0 mix 10X Tris Buffered Saline with Tween 20 TBST 10X 9997 To prepare 1 L 1X TBST add 100 ml 10X TBST to 900 ml dH 0 mix Nonfat Dry Milk 9999 Blocking Buffer 1X TBS with 5 w v nonfat dry milk for 150 ml add 7 5 g nonfat dry milk to 150 ml 1X TBS and mix well Tween 20 should not be present in the Blocking Buffer because it is auto fluorescent and increases non specific background After the blocking step Tween 20 can be reintroduced to subsequent diluent buffers Wash Buffer 1X TBST 0 Bovine Serum Albumin BSA 9998 Primary Antibody Dilution Buffer 1X TBST with 5 BSA or 5 nonfat dry milk as indicated on primary antibody datasheet for 20 ml add 1 0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well 2 Secondary Antibody Dilution Buffer 1X TBST with 5 nonfat dry milk for 20 ml add 1 0 g nonfat dry milk to 20 ml 1X TBST and mix well Secondary antibodies anti rabbit 5151 and 5366 anti mouse 5257 and 5470 3 Prestained Protein Marker Broad Range Premixed Format 7720 4 Blotting Membrane and Paper 12369 This protocol has been optimized for nitrocellulose membranes recommended Pore size 0 2 um is generally recommended a oN o B Protein Blotting A general protocol for sample preparation 1 Treat cells by adding fresh
4. Repeat 2 16 Formaldehyde methanol free 2 Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer 3 Triton X 100 To prepare 50 ml of 0 1 Triton X 100 add 25 ml Triton X 100 to 50 ml 1 X PBS and mix well 3 Incubate for 30 60 min at room temperature 4 50 methanol 4 Wash by centrifugation in 2 3 ml incubation buffer 5 Incubation Buffer Dissolve 0 5 g Bovine Serum Albumin BSA 9998 in 100 ml 1X PBS Store at 4 C 5 Resuspend cells in fluorochrome conjugated secondary antibody diluted in incubation buffer according to the manufacturer s 6 Secondary Antibodies Anti mouse 4408 8890 4410 8887 Anti rabbit 4412 8889 4414 8885 recommendations Anti rat 4416 4418 6 Incubate for 30 min at room temperature 7 Wash by centrifugation in 2 3 ml incubation buffer B Preparation of Whole Blood fixation lysis and permeabilization 8 Resuspend cells in 0 5 ml PBS and analyze on flow cytometer for Immunostaining Reference Chow S Hedley D Grom P Magari R Jacobberger J W Shankey T V 2005 Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte 1 Aliquot 100 ul fresh whole blood per assay tube 2 OPTIONAL Place tubes in rack in 37 C water bath for short term treatments with ligands inhibitors drugs etc 3 Add 65 yl of 10 formaldehyde to each tube
5. 100 l well Cover plate and incubate at 37 C for 1 hr 4 Wash plate Section C Step 3 5 Secondary antibody either streptavidin anti mouse or anti rabbit HRP is diluted 1 1000 in blocking buffer For a single 96 well plate add 10 yl of secondary antibody stock to 9 99 ml of blocking buffer Mix well and add 100 pl well Cover and incubate at 37 C for 30 min 6 Wash plate Section C Step 3 7 Add 100 ul of TMB substrate per well Cover and incubate at 37 C for 10 min 8 Add 100 ul of STOP solution per well Shake gently for a few seconds 9 Read plate on a microplate reader at absorbance 450 nm a Visual Determination Read within 30 min after adding STOP solution b Spectrophotometric Determination Wipe underside of wells with a lint free tissue Read absorbance at 450 nm within 30 min after adding STOP solution A Cell Signaling TECHNOLOGY Notes www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan Chromatin Immunoprecipitation ChIP Enzymatic pg 1 of 3 last updated 8 21 13 Solutions and Reagents Included Reagents Included in SimpleChIP and SimpleChIP Plus Enzymatic Chromatin IP Kits 9002 9003 9004 or 9005 Glycine Solution 10X Buffer A 4X Buffer B 4X ChIP Buffer 10X ChIP Elution Buffer 2X 5 M NaCl 0 5 M EDTA 1 M DTT DNA Binding Reagent A DNA Wash Reagent B DNA Elution Reagent C DNA Sp
6. 2X LumiGLO Reagent and 2X Peroxide 10 Add 50 hl of the detection reagent working solution to each well 11 Use a plate based luminometer set at 425 nm to measure Relative Light Units RLU within 1 10 min following addition of the substrate a Optimal signal intensity is achieved when read within 10 min 08 2013 Cell Signaling Technology Inc PathScan is a registered trademark of Cell Signaling Technology LumiGLO is a registered trademark of Kirkaard and Perry Laboratories www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan PathScan Sandwich ELISA ELISA Antibody Pair last updated 8 21 13 A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 2 Wash Buffer 1X PBS 0 05 Tween 20 20X PBST 9809 3 Blocking Buffer 1X PBS 0 05 Tween 20 1 BSA 4 1X Cell Lysis Buffer 10X Cell Lysis Buffer 9803 To prepare 10 ml of 1X Cell Lysis Buffer add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH20 mix PathScan Sandwich ELISA Lysis Buffer 7018 1X This buffer is ready to use as is Both buf fers can be stored at 4 C for short term use 1 2 weeks Recommended Add 1 mM phenylmethylsulfony fluoride PMSF 8553 immediately before use NOTE Refer to product specific datas
7. ATP 10 mM to 490 ul 1X kinase buffer B Preparing Cell Lysates 1 Aspirate media Treat cells by adding fresh media containing regulator for desired time 2 To harvest cells under nondenaturing conditions remove media and rinse cells once with ice cold 1X PBS 3 Remove PBS and add 0 5 ml ice cold 1X cell lysis buffer to each plate 10 cm and incubate on ice for 5 min 4 Scrape cells off the plate and transfer to microcentrifuge tubes Keep on ice 5 Sonicate on ice three times for 5 sec each 6 Microcentrifuge for 10 min at 4 C 14 000 x g and transfer the supernatant to a new tube The supernatant is the cell lysate If necessary lysate can be stored at 80 C 08 2013 Cell Signaling Technology Inc Sepharose is a registered trademark of GE Healthcare C Immunoprecipitation Cell Lysate Pre Clearing Optional step for unconjugated and biotinylated antibodies 1 Add 10 30 ll of 50 bead slurry either Protein A or G agarose or magnetic beads for unconjugated primary antibodies or 10 ul streptavidin beads 3419 for biotinylated antibodies to 200 ul cell lysate at 1 mg ml 2 Incubate with rotation at 4 C for 30 60 min 3 Microcentrifuge for 10 min at 4 C Transfer the supernatant to a fresh tube 4 Proceed to one of the following specific set of steps depending on the primary antibody used Using Unconjugated Primary Antibodies 1 Add primary antibody at the appropriate dilution a
8. ELISA Kit blue color Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results 3 Add 100 ul of each undiluted or diluted cell lysate to the appropriate well Seal with tape and press firmly onto top of microwells Incubate the plate for 2 hr at 37 C Alternatively the plate can be incubated overnight at 4 C 4 Gently remove the tape and wash wells a Discard plate contents into a receptacle b Wash 4 times with 1X wash buffer 200 ull each time per well c For each wash strike plates on fresh paper towels hard enough to remove the residual solution in each well but do not allow wells to completely dry at any time d Clean the underside of all wells with a lint free tissue 5 Add 100 ul of detection antibody green color to each well Seal with tape and incubate the plate at 37 C for 1 hr 6 Repeat wash procedure Section C Step 4 7 Add 100 ul of HRP linked secondary antibody red color to each well Seal with tape and incubate the plate for 30 min at 37 C 8 Repeat wash procedure Section C Step 4 9 Add 100 ul of TMB substrate to each well Seal with tape and incubate the plate for 10 min at 37 C or 30 min at 25 C 10 Add 100 ul of STOP solution to each well Shake gently for a few seconds NOTE Initial color of positive reaction is blue which changes to yellow upon addition of STOP solution 11 Read results a Visual D
9. heavy chains For proteins with molecular weights near 25 kDa Mouse Anti rabbit IgG Conformation Specific L27A9 mAb 3678 or Mouse Anti rabbit IgG Conformation Specific L27A9 mAb HRP Conjugate 5127 is recommended For Analysis by Kinase Assay 1 Wash pellet twice with 500 pl 1X kinase buffer Keep on ice 2 Suspend pellet in 40 ul 1X kinase buffer supplemented with 200 uM ATP and appropriate substrate 3 Incubate for 30 min at 30 C 4 Terminate reaction with 20 ul 3X SDS sample buffer Vortex then microcentrifuge for 30 sec 5 Transfer supernatant containing phosphorylated substrate to another tube 6 Heat the sample to 95 100 C for 2 5 min and microcentrifuge for 1 min at 14 000 x g 7 Load the sample 15 30 ll on SDS PAGE 4 20 Immunohistochemistry IHC Paraffin using SignalStain Boost Detection Reagent last updated 8 21 13 IMPORTANT Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine whether a product is validated and approved for use on paraffin embedded IHC P tissue sections Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 Xylene 2 Ethanol anhydrous denatured histological grade 100 and 95 3 Deionized water dH 0 4 Hematoxylin opt
10. media containing regulator for desired time 2 Aspirate media from cultures wash cells with cold 1X PBS aspirate 3 Lyse cells by adding 1X SDS sample buffer 100 pl per well of 6 well plate or 500 ul per plate of 10 cm diameter plate Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube Keep on ice 4 Sonicate for 10 15 sec to complete cell lysis and shear DNA to reduce sample viscosity 5 Heat a 20 pl sample to 95 100 C for 5 min cool on ice 6 Microcentrifuge for 5 min 7 Load 20 ul onto SDS PAGE gel 10 cm x 10 cm NOTE Loading of prestained molecular weight markers 7720 10 pl lane is recommended to verify electrotransfer and to determine molecular weights Prestained markers are autofluorescent at near infrared wavelengths 8 Electrotransfer 0 nitrocellulose membrane 12369 C Membrane Blocking and Antibody Incubations NOTE Volumes are 1 Optional After transfer wash nitrocell 2 Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature CRITICAL STEP Do not include Tween 20 in blocking buffer Se 3 Wash three times ction A Step 8 or 10 cm x 10 cm 100 cm of membrane for different sized membranes adjust volumes accordingly lose membrane with 25 ml TBS for 5 min at room temperature or 5 min each with 15 ml of TBST 4 Incubate membrane and primary antibody at the appropriate dilution as recommended in the product datash
11. plate of cells prior to cross linking to determine accurate cell number Add more tissue or cells or less micrococcal nuclease to the chromatin digest See Section B of troubleshooting guide for optimization of chromatin digestion No product or very little product in the input PCR reactions Not enough DNA added to the PCR reaction or conditions are not optimal PCR amplified region may span nucleosome free region Not enough chromatin added to the IP or chromatin is over digested Add more DNA to the PCR reaction or increase the number of amplification cycles Optimize the PCR conditions for experimental primer set using purified DNA from cross linked and digested chromatin Design a different primer set and decrease length of amplicon to less than 150 bp see primer design recommendations in Protocol Section VIII For optimal ChIP results add 5 to 10 ug chromatin per IP No product in the positive control histone H3 IP RPL30 PCR reaction Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short Incomplete elution of chromatin from Protein G beads Be sure to add 5 to 10 hg of chromatin and 10 ul of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G beads Elution of chromatin from Protein G beads is optimal at 65 C with frequent mixing to keep beads suspended in solution Quantity of product in the negativ
12. plates compatible with the model of PCR machine to be used PCR reactions should include the positive control histone H3 sample the negative control normal rabbit IgG sample a tube with no DNA to control for contamination and a serial dilution of the 2 input chromatin DNA undiluted 1 5 1 25 1 125 to create a standard curve and determine the efficiency of amplification 2 Add 2 ul of the appropriate DNA sample to each tube or well of the PCR plate 3 Prepare a master reaction mix as described below Add enough reagents for two extra reactions to account for loss of volume Add 18 ul of reaction mix to each PCR reaction tube or well Reagent Volume for 1 PCR Reaction 18 pI Nuclease free H O 6 yl 5 uM RPL30 Primers 2 pl 2X SYBR Green Reaction Mix 10 ul 4 Start the following PCR reaction program a Initial denaturation 95 C 3 min b Denature 95 C 15 sec c Anneal and extension 60 C 60 sec d Repeat steps b and for a total of 40 cycles 5 Analyze quantitative PCR results using the software provided with the real time PCR machine Alternatively one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below With this method signals obtained from each IP are expressed as a percent of the total input chromatin Percent Input 2 X 2C0 2 Input Sample C T IP Sample CT Cr Threshold cycle of PCR reaction ia Cell Signaling TECHNOLOGY Notes www cel
13. stain 100 of the cases of a given indication It is possible that the sample is truly negative HIGH BACKGROUND Cause Solution Slide preparation Inadequate deparaffinization may cause spotty uneven background staining Repeat the experiment with new sections using fresh xylene Peroxidase quenching Endogenous peroxidase activity in samples may produce excess background signal if an HRP based detection system is being used Quench slides in a 3 H202 solution diluted in RODI water for 10 min prior to incubation with the primary antibody Biotin block Using biotin based detection systems with samples that have high levels of endogenous biotin such as kidney and liver tissues may be problematic In this case use a polymer based detection system such as SignalStain Boost IHC Detection Reagents 8114 and 8125 A biotin block may also be performed after the normal blocking procedure prior to incubation in primary antibody Blocking Block slides with 1X TBS 9997 with 5 Normal Goat Serum 5425 for 30 min prior to incubation with the primary antibody Antibody dilution diluent Consult CST product datasheet for the recommended dilution and diluent Titration of the antibody may be required if a reagent other than the one recommended is used Secondary cross reactivity The secondary antibody may bind endogenous IgG causing high background in some samples where the secondary antibody is raised in the same species as the sample being tes
14. the positive o length of approximately 150 900 bp Mix nar a ae min The NOTE For optimal ChIP results it is highly critical that the chromatin is of appropri control Histone H3 D2B12 XP Rabbit mAb add 10 pl to the IP sample For amount of micrococcal nuclease required to digest DNA to the optimal length ate size and concentration Over digestion of chromatin may diminish signal in the the negative control Normal Rabbit IgG add 1 pl 1 po to 2 pl 2 po to the IP may need to be determined empirically for individual tissues and cell lines see PCR quantification Under digestion of chromatin may lead to increased background sample Incubate IP samples 4 hr to overnight at 4 C with rotation Troubleshooting Guide Section B HeLa nuclei digested with 0 5 ul micrococcal signal and lower resolution Adding too little chromatin to the IP may result in 5 a Resuspend ChiP Grade Protein G Agarose Beads by gently vortexing Immedi nuclease per 4 x 108 calls andimolge liver tissue digested with 0 5 ul micrococ diminished signal in the PCR quantification A protocol for optimization of chromatin ately add 30 ul of Protein G Agarose Beads to each IP reaction and incubate cal nuclease per 25 mg of tissue gave the appropriate length DNA fragments digestion can be found in the Troubleshooting Guide for 2 hr at 4 C with rotation 4 Stop digest by adding 10 pl of 0 5 M EDTA per IP prep and placing tube on ice V Chromatin IP b E As aia aan ae and 5 Pellet nuc
15. time Film exposure times of more than 30 sec lead to increased background signal To avoid long exposure times it is important to use cell lines or tissues with adequate protein expression levels and use recommended primary antibody incubation conditions see primary antibody dilution and or incubation section If necessary use treatment to induce expression or modification LOW SIGNAL The protein of interest cannot be detected after a short exposure of blot to film Cause Solution Lysate preparation 20 30 hg total protein from whole cell extracts per lane is usually sufficient for detection If basal levels of target protein or protein modification are low it may be necessary to induce expression or modification via chemical stimulant You may want to investigate alternative cell lines or tissues in which the protein of interest is more abundant Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors for phospho targets Phosphatase Inhibitor Cocktail 100X 5870 Protease Phosphatase Inhibitor Cocktail 100X 5872 Protease Cocktail 5871 Lysates should always be sonicated to ensure efficient protein extraction of chromatin and membrane bound targets Please visit the Controls Table on our website for suggested positive controls and treatments Primary antibody dilution Incubate primary antibody overnight at 4 C in TBST at the recommended dilution with the recommended blocking a
16. times in PBS for 5 min each d Proceed with Immunostaining Section C C Immunostaining NOTE All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light tight box or covered dish plate to prevent drying and fluorochrome fading 1 Block specimen in blocking buffer for 60 min 2 While blocking prepare primary antibody by diluting as indicated on datasheet in antibody dilution buffer 3 Aspirate blocking solution apply diluted primary antibody 4 Incubate overnight at 4 C 5 Rinse three times in 1X PBS for 5 min each NOTE If using a fluorochrome conjugated primary antibody then skip to Section C Step 8 6 Incubate specimen in fluorochrome conjugated secondary antibody diluted in antibody dilution buffer for 1 2 hr at room temperature in the dark 7 Rinse three times in 1X PBS for 5 min each 8 Coverslip slides with Prolong Gold Antifade Reagent 9071 or Prolong Gold Antifade Reagent with DAPI 8961 9 For best results allow mountant to cure overnight at room temperature For long term storage store slides flat at 4 C protected from light 08 2013 Cell Signaling Technology Inc CST is a trademark of Cell Signaling Technology Prolong is a registered trademark of Molecular Probes Inc Triton X 100 is a trademark of The Dow Chemical Company A Cell Signaling TECHNOLOGY Notes www cellsignal com support USA amp Europe www cst c c
17. DI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix NOTE Adjust pH to 8 0 2 Formaldehyde 16 methanol free Polysciences Inc cat 18814 use fresh store opened vials at 4 C in dark Dilute 1 in 4 in 1X PBS to make a 4 formaldehyde solution 3 Blocking Buffer 1X PBS 5 normal serum 0 3 Triton X 100 To prepare 10 ml add 0 5 ml normal serum from the same species as the secondary antibody e g Normal Goat Serum 5425 to 9 ml 1X PBS and mix well While stirring add 30 ul Triton X 100 4 Antibody Dilution Buffer 1X PBS 1 BSA 0 3 Triton X 100 To prepare 10 ml add 30 pl Triton X 100 to 10 ml 1X PBS Mix well then add 0 1g BSA 9998 mix 5 Fluorochrome conjugated Secondary Antibodies Anti mouse 4408 4409 8890 4410 Anti rabbit 4412 4413 8889 4414 Anti rat 4416 4417 4418 6 Prolong Gold Antifade Reagent 9071 Prolong Gold Antifade Reagent with DAPI 8961 Reagents specific to IF P application 1 Xylene 2 Ethanol anhydrous denatured histological grade 100 and 95 3 Antigen Unmasking a For Citrate 10 mM Sodium Citrate Buffer To prepare 1 L add 2 94 g CsH Na 07 2H 0 to 1 L dH 0 Adjust pH to 6 0 b For EDTA 1 mM EDTA To prepare 1 L add 0 372 g EDTA C H N OgNa e2H 0 to 1 L dH 0 Adjust pH to 8 0 B Specimen Preparation NOTE Cells should be grown trea
18. REFERENCE MATERIAL ia Cell Signaling TECHNOLOGY EIRENE Reagents Protocole Cell Signaling Technology approaches validation from the perspective of the scientist because that is who we are We believe it takes more than just seeing a band at the expected molecular weight to validate antibody specificity Our scientists validate all of the products from CST in house for quality and specificity across broad applications The result is a set of optimized protocols tested with every product Using CST validated products with CST validated protocols provides reproducible and reliable results And just in case an unexpected result arises the same scientists who validate our reagents and use these protocols every day are available to provide you with technical support www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan For western blots incubate membrane with diluted primary antibody in either 5 w v BSA or nonfat dry milk 1X TBS 0 1 Tween 20 at 4 C with gentle shaking overnight NOTE Please refer to primary antibody datasheet or product webpage for recommended primary antibody dilution buffer and recommended antibody dilution A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 2 10X Tri
19. agent and 20X Peroxide 7003 B Preparing Cell Lysates For adherent cells 1 Aspirate media when the culture reaches 80 90 confluence Treat cells by adding fresh media containing regulator for desired time 2 Remove media and rinse cells once with ice cold 1X PBS 3 Remove PBS and add 0 5 ml ice cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate 10 cm diameter and incubate the plate on ice for 5 min 4 Scrape cells off the plate and transfer to an appropriate tube Keep on ice 5 Sonicate lysates on ice 6 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in single use aliquots For suspension cells 1 Remove media by low speed centrifugation 1 200 rpm when the culture reaches 0 5 1 0 x 10 viable cells ml Treat cells by adding fresh media containing regulator for desired time 2 Collect cells by low speed centrifugation 1 200 rpm and wash once with 5 10 ml ice cold 1X PBS 3 Cells harvested from 50 ml of growth medium can be lysed in 2 0 ml of 1X cell lysis buffer plus 1 mM PMSF 4 Sonicate lysates on ice 5 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in single use aliquots C Test Procedure 1 After the microwell strips have reached room temperature break off the required number of microwel
20. and store on ice Disaggregate tissue into single cell suspension using a Medimachine Section B or Dounce homogenizer Section C B Tissue Disaggregation Using Medimachine from BD Biosciences part 340587 1 Cut off the end of a 1 ml pipette tip to enlarge the opening for transfer of tissue chunks 2 Transfer 1 ml of tissue resuspended in 1X PBS PIC into the top chamber of a 50 mm medicone Part 340592 3 Grind tissue for 2 min according to manufacturer s instructions 4 Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle Transfer cell suspension to a 15 ml conical tube and place on ice 5 Repeat steps 2 4 until all the tissue is processed into a homogenous suspension 6 If more grinding is necessary add more 1X PBS PIC to tissue Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension 7 Check for single cell suspension by microscope optional 8 Centrifuge cells at 1 500 rpm in a bench top centrifuge for 5 min at 4 C 9 Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion Section Ill C Tissue Disaggregation Using a Dounce Homogenizer 1 Transfer tissue resuspended in 1X PBS PIC to a Dounce homogenizer 2 Disaggregate tissue pieces with 20 25 strokes Check for single cell suspen sion by microscope optional 3 Transfer cell suspension to a 15 ml conical t
21. ceed with detection Section D on For Biotinylated Primary Antibodies 1 Incubate membrane and primary antibody at the appropriate dilution as recom mended in the product datasheet in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4 C Wash three times for 5 min each with 15 ml of TBST Incubate membrane with Streptavidin HRP 3999 at the appropriate dilution in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature 4 Wash three times for 5 min each with 15 ml of TBST 5 Proceed with detection Section D Do not add Anti biotin HRP linked Antibody for detection of biotinylated protein markers There is no need The Streptavidin HAP will also visualize the biotinylated markers on D Detection of Proteins 1 Incubate membrane with 10 ml LumiGLO 0 5 ml 20X LumiGLO 7003 0 5 ml 20X Peroxide and 9 0 ml purified water or 10 ml SignalFire 6883 5 ml Reagent A 5 ml Reagent B with gentle agitation for 1 min at room temperature 2 Drain membrane of excess developing solution do not let dry wrap in plastic wrap and expose to x ray film An initial 10 sec exposure should indicate the proper exposure time NOTE Due to the kinetics of the detection reaction signal is most intense immediately following incubation and declines over the following 2 hr 08 2013 Cell Signaling Technology Inc Phototope and SignalFire are trademarks of Cell Signaling Technolog
22. cross linked chromatin preparation Section Ill Step 3 Purify DNA from samples using DNA purification spin columns as described in 9 per IP For example for 10 IPs prepare a tube containing 4 ml 1X ChIP Buffer i Pe ripe i a PIC per IP prep Incubate on Section VII 400 pl 10X ChIP buffer 3 6 ml water 20 ul 200X PIC 1 ml digested ee en 4 After purification of DNA remove a 10 ul sample and determine DNA fragment chromatin preparation J n ei by en 3 000 la i A ie Uta i 5 mra size by electrophoresis on a 1 agarose gel with a 100 bp DNA marker DNA 3 Remove a 10 pl sample of the diluted chromatin and transfer to a microfuge af ately el s4 ee sara le Sta se ae n should be digested to a length of approximately 150 900 bp 1 6 nucleosomes tube This is your 2 input sample which can be stored at 20 C until further ab iB fe g DTT per t prep Transfer oa toa 1 5 ml fie erie 5 To determine DNA concentration transfer 2 ul of purified DNA to 98 ul use Step 1 in Section VI ube up to 1 ml total per tube nuclease free water to give a 50 fold dilution and read the 0Dzeo The 4 For each IP transfer 500 ul of the diluted chromatin to a 1 5 ml microcentri i concentration of DNA in ig ml is OD2so x 2 500 DNA concentration should fuge tube and add the immunoprecipitating antibody The amount of antibody a sae ya l Ea Dre h a rral ideally be between 50 and 200 pg ml required per IP varies and should be determined by the user For
23. d after sonication preparation for IP of chromatin complexes 7 Wash Protein G Beads by adding 1 ml of low salt wash to the beads and incubate Batnre starting at 4 C for 5 min with rotation Repeat Steps 6 and 7 two additional times for a HeLa nuclei were completely lysed after 3 sets of 20 sec pulses using a VirTis g z A total of 3 low salt washes Virsonic 100 Ultrasonic Homogenizer Sonicator at setting 6 with a 1 8 inch e Remove and warm 200X Protease Inhibitor Cocktail PIC Make sure PIC is 8 Add 1 ml of high salt wash to the beads and incubate at 4 C for 5 min with probe Alternatively nuclei can be lysed by homogenizing the lysate 20 times in completely thawed rotation a Dounce homogenizer however lysis may not be as complete e Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution 9 a Pellet Protein G Agarose Beads in each IP by brief 1 min centrifugation at 8 Clarify lysates by centrifugation at 10 000 rpm in a microcentrifuge for 10 min e Thaw digested chromatin preparation Section Ill Step 9 and place on ice 6 000 rpm in a microcentrifuge Remove supernatant and immediately at 4 C e Prepare low salt wash 3 ml 1X ChIP buffer 300 pl 10X ChIP buffer 2 7 ml H20 proceed to Section VI 9 Transfer supernatant to a new tube This is the cross linked chromatin prepara per IP prep Store at room temperature until use b Pellet Protein G Magnetic Beads in each IP by placing the tubes in a Magnetic tion which sh
24. dy resuspend cells in 0 5 ml 1X PBS and analyze on flow cytometer for unconjugated or biotinylated primary antibodies proceed to immunostaining Step 9 9 Resuspend cells in fluorochrome conjugated secondary antibody or fluorochrome conjugated avidin diluted in incubation buffer at the recommended dilution 10 Incubate for 30 min at room temperature 11 Wash by centrifugation in 2 3 ml incubation buffer 12 Resuspend cells in 0 5 ml PBS and analyze on flow cytometer alternatively for DNA staining proceed to optional DNA stain Section E E Optional DNA Dye 1 Resuspend cells in 0 5 ml of DNA dye e g Propidium lodide Pl RNase Staining Solution 4087 2 Incubate for at least 30 min at room temperature 3 Analyze cells in DNA staining solution on flow cytometer ia Cell Signaling TECHNOLOGY Flow Cytometry F last updated 8 21 13 Alternate for combined staining of intracullular proteins and cell surface markers in blood A Solutions and Reagents C Staining Using Unlabeled Primary and Conjugated Secondary Antibodies NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water NOTE Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 1 Add 2 3 ml incubation buffer to each tube and rinse by centrifugation
25. e control Rabbit IgG IP and positive control histone H3 IP PCR reactions is equivalent high background signal Too much or not enough chromatin added to the IP reaction Alternatively too much antibody added to the IP reaction Too much DNA added to the PCR reaction or too many cycles of amplification For optimal ChIP results add 5 to 10 ug of chromatin and 10 ul of histone H3 antibody to each IP reac tion Reduce the amount of normal rabbit IgG to 1 ul per IP Add less DNA to the PCR reaction or decrease the number of PCR cycles It is very important that the PCR products are analyzed within the linear amplification phase of PCR Otherwise the differences in quantities of starting DNA cannot be accurately measured Alternatively quantify immunoprecipitations using real time quantitative PCR No product in the Experimental Antibody IP PCR reaction www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan Not enough DNA added to the PCR reaction Not enough antibody added to the IP reaction Antibody does not work for ChIP Add more DNA to the PCR reaction or increase the number of amplification cycles Typically a range of 1 to 5 ug of antibody are added to the IP reaction however the exact amount depends greatly on the individual antibody Increase the amount of antibody added to the IP Find an alternate antibody source i our mission To deli
26. e the use of a pressure cooker Unmasking retrieval buffer Staining of particular tissues or antigen targets may require an optimized unmasking buffer Refer to product datasheet for antigen unmasking buffer recommendations Always prepare fresh 1X solutions daily Antibody dilution diluent Consult CST product datasheet for the recommended dilution and diluent Titration of the antibody may be required if a reagent other than the one recommended is used Incubation time Primary antibody incubation according to a rigorously tested protocol provides consistent reliable results CST antibodies have been developed and validated for optimal results when incubated overnight at 4 C Detection system Polymer based detection reagents such as SignalStain Boost IHC Detection Reagents 8114 and 8125 in conjunction with SignalStain DAB Substrate Kit 8059 are more sensitive than avidin biotin based detection systems Standard secondary antibodies directly conjugated with HRP may not provide sufficient signal amplification Always verify the expiration date of the detection reagent prior to use Negative staining A complete lack of staining may indicate an issue with the antibody or protocol Employ a high expressing positive control such as paraffin embedded cell pellets to ensure that the antibody and procedure are working as expected Phospho specific antibodies in particular or any antibody directed against a rarely expressed protein may not
27. eet in 10 ml primary antibody di or 5 min each with 15 ml of TBST e with fluorophore conjugated secondary antibody 5470 5257 5366 5151 1 5000 1 25 000 stock in 10 ml of secondary antibody dilution buffer with gentle agitation for 1 hr at room temperature or 5 min each with 15 ml of TBST 5 Wash three times 6 Incubate membran dilution of 1 mg m 7 Wash three times D Detection of lution buffer with ge Proteins 1 Drain membrane 0 excess TBST and a 2 Scan membrane using an appropriate fi ntle agitation overnight at 4 C low to dry CRITICAL STEP Membrane must be dry for fluorescent staining uorescent scanner following the manufacturer s recommendations 08 2013 Cell Signaling Technology Inc Tween 20 is a registered trademark of ICI Americas Inc www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan HIGH BACKGROUND General background is high or nonspecific bands appear after a short exposure of blot to film Be conscious of the fact that motif antibodies post translational modifications and splice variants may result in multiple banding Lysate preparation Freshly prepared samples result in fewer nonspecific and degradation bands and therefore yield a cleaner blot In general tissue extracts tend to contain more background bands and degradation products than cell line extracts due to connective tissue Using fresh so
28. entration Section IV Before starting e Remove and warm 200X Protease Inhibitor Cocktail PIC and 10X glycine solution Make sure PIC is completely thawed e Prepare 3 ml of 1X Phosphate Buffered Saline PBS 15 ul 200X PIC per 25 mg of tissue to be processed and place on ice e Prepare 45 ul of 37 formaldehyde per 25 mg of tissue to be processed and keep at room temperature Use fresh formaldehyde that is not past the manufacturer s expiration date A Cross linking 1 Weigh the fresh or frozen tissue sample Use 25 mg of tissue for each IP to be performed 2 Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade Keep dish on ice It is important to keep the tissue cold to avoid protein degradation 3 Transfer minced tissue to a 15 ml conical tube 4 Add 1 ml of 1X PBS PIC per 25 mg tissue to the conical tube 5 To crosslink proteins to DNA add 45 pl of 37 formaldehyde per 1 ml of 1X PBS PIC and rock at room temp for 20 min Final formaldehyde concentration is 1 5 6 Stop cross linking by adding 100 ul of 10X glycine per 1 ml of 1X PBS PIC and mix for 5 min at room temperature 7 Centrifuge tissue at 1 500 rpm in a bench top centrifuge for 5 min at 4 C 8 Remove supernatant and wash with 1 ml 1X PBS PIC per 25 mg tissue 9 Repeat centrifugation Section A Step 7 10 Remove supernatant and resuspend tissue in 1 ml 1X PBS PIC per 25 mg tissue
29. epare cross linked nuclei from 125 mg of tissue or 2 X 10 cells equivalent of 5 IP preps as described in Protocol micrococcal nuclease in each digest accordingly Alternatively the digestion time can be changed to increase or decrease Sections Il and Ill Stop after Step 2 of Protocol Section Ill and proceed as described below the extent of DNA fragmentation 2 Transfer 100 ul of the nuclei preparation into 5 individual 1 5 ml microcentrifuge tubes and place on ice 3 Add 3 ul micrococcal nuclease stock to 27 ul of 1X Buffer B DTT 1 10 dilution of enzyme 08 2013 Cell Signaling Technology Inc SimpleChIP and XP are registered trademarks of Cell Signaling Technology SYBR Green and Prolong are registered trademarks of Molecular Probes Inc Trizma is a trademark of Sigma Chemical Company BD is a trademark of BD Biosciences ia Cell Signaling TECHNOLOGY Chromatin Immunoprecipitation ChIP Troubleshooting Guide pg 2 of 2 Problem Concentration of the digested chromatin is too low low chromatin yield Possible Causes Not enough tissue or cells were added to the chromatin digestion or cell nuclei were not completely lysed after digestion last updated 8 21 13 Recommendation Add additional chromatin to each IP to give at least 5 Hg IP and continue with protocol Weigh tissue or count a separate plate of cells prior to cross linking to determine accurate cell number Some tissues may requi
30. er two times for 5 min Incubate for 10 min at room temperature in methanol peroxidase Wash sections in wash buffer two times for 5 min Block each section with 100 400 ul blocking solution for 1 hr at room temperature Remove blocking solution and add 100 400 ul primary antibody diluted in blocking solution to each section Incubate overnight at 4 C Equilibrate SignalStain Boost Detection Reagent to room temperature Remove antibody solution and wash sections in wash buffer three times for 5 min each Cover section with 1 3 drops SignalStain Boost Detection Reagent as needed Incubate in a humidified chamber for 30 min at room temperature Wash sections three times with wash buffer for 5 min each Add 1 drop 30 ul SignalStain DAB Chromogen Concentrate to 1 ml SignalStain DAB Diluent and mix well before use Apply 100 400 yl SignalStain DAB to each section and monitor closely 1 10 min generally provides an acceptable staining intensity Immerse slides in dH 0 If desired counterstain sections with hematoxylin per manufacturer s instructions Wash sections in dH O two times for 5 min each Dehydrate sections a Incubate sections in 95 ethanol two times for 10 sec each b Repeat in 100 ethanol incubating sections two times for 10 sec each c Repeat in xylene incubating sections two times for 10 sec each Mount sections with coverslips www cellsignal com support USA amp Europe w
31. etermination Read within 30 min after adding STOP solution b Spectrophotometric Determination Wipe underside of wells with a lint free tissue Read absorbance at 450 nm within 30 min after adding STOP solution A Cell Signaling TECHNOLOGY PathScan Sandwich ELISA ELISA Chemiluminescent last updated 8 21 13 NOTE Refer to product specific datasheets or product webpage for assay incubation temperature This chemiluminescent ELISA is offered in low volume microplate Samples and reagents only require 50 pl per microwell A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 10X PBS to 950 ml dH 0 mix 2 Bring all microwell strips to room temperature before use 3 Prepare 1X wash buffer by diluting 20X Wash Buffer included in each PathScan Sandwich ELISA Kit in dH 0 4 1X Cell Lysis Buffer 10X Cell Lysis Buffer 9803 To prepare 10 ml of 1X Cell Lysis Buffer add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH 0 mix PathScan Sandwich ELISA Lysis Buffer 7018 1X This buffer is ready to use as is Both buf fers can be stored at 4 C for short term use 1 2 weeks Recommended Add 1 mM phenylmethylsulfony fluoride PMSF 8553 immediately before use NOTE Refer to product specific datasheet or webpage for lysis buffer recommendation 5 20X LumiGLO Re
32. gent 5 BSA or 5 nonfat dry milk The use of alternate blocking agents such as gelatin serum protein free and or incubation blocking agents casein or mixed blocking agents may reduce target signal intensily For optimal results with individual antibodies please consult the product datasheet for recommended dilution buffer SDS PAGE Gel selection In general Tris Glycine gels are recommended However for high molecular weight proteins we recommend Tris Acetate gels and associated buffers Proteins transfer more efficiently from 1 mm gels than from 1 5 mm gels The use of thicker gels can result in incomplete transfer of high molecular weight proteins so we recommend monitoring transfer efficiency and implementing modifications of transfer conditions to optimize for the size of your protein target of interest Transfer and Membrane Incomplete transfer can be corrected by longer transfer or by the use of higher voltage In general we recommend including 20 methanol in transfer buffer and performing a wet transfer for 1 5 hr at 70 V For high molecular weight proteins we recommend reducing the methanol in transfer buffer to 5 to improve transfer efficiency and avoid fixing large proteins in the gel matrix as well as increasing transfer time to 3 hr at 70 V Over transfer protein transfer through membrane can be problematic when examining smaller proteins For small proteins it is important to use a 0 2 um pore size membrane a
33. heet or webpage for lysis buffer recommendation 5 Bovine Serum Albumin BSA 9998 6 TMB Substrate 7004 7 STOP Solution 7002 NOTE Reagents should be made fresh daily B Preparing Cell Lysates For adherent cells 1 Aspirate media when the culture reaches 80 90 confluence Treat cells by adding fresh media containing regulator for desired time 2 Remove media and rinse cells once with ice cold 1X PBS 3 Remove PBS and add 0 5 ml ice cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate 10 cm diameter and incubate the plate on ice for 5 min 4 Scrape cells off the plate and transfer to an appropriate tube Keep on ice 5 Sonicate lysates on ice 6 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in single use aliquots For suspension cells 1 Remove media by low speed centrifugation 1 200 rpm when the culture reaches 0 5 1 0 x 10 viable cells ml Treat cells by adding fresh media containing regulator for desired time 2 Collect cells by low speed centrifugation 1 200 rpm and wash once with 5 10 ml ice cold 1X PBS 3 Cells harvested from 50 ml of growth media can be lysed in 2 0 ml of 1X cell lysis buffer plus 1 mM PMSF 4 Sonicate lysates on ice 5 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in
34. in Col umns Protease Inhibitor Cocktail 200X RNAse A 10 mg ml Micrococcal Nuclease 2000 gel units pl Proteinase K 20 mg ml SimpleChIP Human RPL30 Exon 3 Primers 7014 SimpleChIP Mouse RPL30 Intron 2 Primers 7015 Histone H3 D2B12 XP Rabbit mAb ChIP Formulated 4620 Normal Rabbit IgG 2729 ChiP Grade Protein G Magnetic Beads 9006 or ChiP Grade Protein G Agarose Beads 9007 Reagents Not Included Formaldehyde 37 Ethanol 96 100 Isopropanol 1X PBS 20X PBS 9808 Nuclease free water Taq DNA polymerase dNTP Mix For Kits 9002 and 9003 only PMSF 8553 0 1 M stock For Kits 9003 and 9005 only 6 Tube Magnetic Separation Rack 7017 l Tissue Cross linking and Sample Preparation IMPORTANT Section exclusive to use of SimpleChIP Plus Kits 9004 amp 9005 When harvesting tissue remove unwanted material such as fat and necrotic material from the sample Tissue can then be processed and cross linked immediately or fro zen on dry ice for processing later For optimal chromatin yield and ChIP results use 25 mg of tissue for each IP to be performed The chromatin yield does vary between tissue types and some tissues may require more than 25 mg for each IP Please see Troubleshooting Guide Section A for more information regarding the expected chromatin yield for different types of tissue One additional chromatin sample should be processed for Analysis of Chromatin Digestion and Conc
35. in centrifugation at 6 000 rpm in a microcentrifuge b Pellet Protein G Magnetic Beads by placing the tubes in a Magnetic Separation Rack and wait 1 to 2 min for solution to clear Carefully transfer eluted chromatin supernatant to a new tube To all tubes including the 2 input sample from Step 1 reverse cross links by adding 6 ul 5 M NaCl and 2 ul Proteinase K and incubate 2 hr at 65 C This incubation can be extended overnight Immediately proceed to Section VII Alternatively samples can be stored at 20 C However to avoid formation of a precipitate be sure to warm samples to room temperature before adding DNA binding reagent A Section VII Step 1 ea N VII DNA Purification Using Spin Columns Before starting e Add 12 ml of isopropanol to DNA binding reagent A and 24 ml of ethanol 96 100 to DNA wash reagent B before use These steps only have to be performed once prior to the first set of DNA purifications e Remove one DNA purification spin column and collection tube for each DNA sample Section VI 1 Add 600 ul DNA binding reagent A to each DNA sample and vortex briefly 4 volumes of DNA binding reagent A should be used for every 1 volume of sample 2 Transfer 375 ul of each sample from Step 1 to a DNA purification spin column in collection tube 3 Centrifuge at 14 000 rpm in a microcentrifuge for 30 sec 4 Remove spin column from the collection tube and discard the liquid Replace spin column in
36. ional 5 Wash Buffer a 10X Tris Buffered Saline with Tween 20 TBST 9997 To prepare 1 L 1X TBST add 100 ml 10X TBST to 900 ml dH 0 mix 0 6 Antibody Diluent Options a SignalStain Antibody Diluent 8112 b TBST 5 normal goat serum To 5 ml 1X TBST add 250 pl Normal Goat Serum 5425 c PBST 5 normal goat serum To 5 ml 1X PBST add 250 hl Normal Goat Serum 5425 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH20 mix 1X PBS 0 1 Tween 20 1X PBST To prepare 1 L 1X PBST add 100 ml 10X PBS to 900 ml dH20 Add 1 ml Tween 20 and mix 7 Antigen Unmasking Options a Citrate 10 mM Sodium Citrate Buffer To prepare 1 L add 2 94 g sodium citrate trisodium salt dihydrate C H Na 0 2H20 to 1 L dH20 Adjust pH to 6 0 b EDTA 1 mM EDTA To prepare 1 L add 0 372 g EDTA C H N 0 Na 2H20 to 1 L dH20 Adjust pH to 8 0 c TE 10 mM Tris 1 mM EDTA pH 9 0 To prepare 1 L add 1 21 g Tris base C H NO and 0 372 g EDTA C H N 0 Na 2H 0 to 950 ml dH 0 Adjust pH to 9 0 then adjust final volume to 1 L with dH20 d Pepsin 1 mg ml in Tris HCl pH 2 0 8 3 Hydrogen Peroxide To prepare 100 ml add 10 ml 30 H 0 to 90 ml dH 0 9 Blocking Solution TBST 5 Normal Goat Serum to 5 ml 1X TBST add 250 ul Normal Goat Serum 5425 10 Detection System SignalStain Boost IHC Detection Reagents HRP Mouse 8125 HAP Rabbit 8114 11 Substrate SignalStain
37. it 7074 anti mouse 7076 16 Detection Reagent LumiGLO chemiluminescent reagent and peroxide 7003 or SignalFire ECL Reagent 6883 9 D on B Protein Blotting A general protocol for sample preparation 1 Treat cells by adding fresh media containing regulator for desired time 2 Aspirate media from cultures wash cells with 1X PBS aspirate 3 Lyse cells by adding 1X SDS sample buffer 100 ul per well of 6 well plate or 500 ul for a 10 cm diameter plate Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube Keep on ice 4 Sonicate for 10 15 sec to complete cell lysis and shear DNA to reduce sample viscosity 5 Heat a 20 ul sample to 95 100 C for 5 min cool on ice Microcentrifuge for 5 min 7 Load 20 HI onto SDS PAGE gel 10 cm x 10 cm NOTE Loading of prestained molecular weight markers 7720 10 l lane to verify electrotransfer and biotinylated protein ladder 7727 10 ul lane to determine molecular weights are recommended Electrotransfer to nitrocellulose membrane 12369 o 2 C Membrane Blocking and Antibody Incubations NOTE Volumes are for 10 cm x 10 cm 100 cm of membrane for different sized membranes adjust volumes accordingly I Membrane Blocking For HRP Conjugated Primary Antibodies 1 Optional After transfer wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature 2 Incuba
38. ks and processed chlorine free EUROPE MIDDLE EAST amp AFRICA Tel 31 0 71 568 1060 E mail eusupport cellsignal eu www cellsignal com JAPAN AUTHORIZED DISTRIBUTORS Tel 03 3295 1630 See a full list of our distributors online Support info cstj co jp www cellsignal com Distributors www cstj co jp FRONT COVER IMAGE 6 Tubulin 9F3 Rabbit mAb Alexa Fluor 555 Conjugate 2116 Confocal IF analysis of Drosophila egg chambers using 2116
39. lei by centrifugation at 13 000 rpm in a microcentrifuge for 1 min at For optimal ChIP results use approximately 5 to 10 bg of digested cross linked incubate for 2 hr at 4 C with rotation 4 C and remove supernatant chromatin as determined in Section IV per IP This should be roughly equivalent to 6 a Pellet Protein G Agarose Beads in each IP by brief 1 min centrifugation at 6 Resuspend nuclear pellet in 100 pil of 1X ChIP buffer PIC per IP prep and a single 100 pl IP prep from 25 mg of disaggregated tissue or 4 x 10 tissue culture 6 000 rpm in a microcentrifuge and remove supernatant incubate on ice for 10 min cells Typically 100 hl of digested chromatin is diluted into 400 yl 1X ChIP Buffer Tan ennai ean Se Saar ane von rere prior to the addition of antibodies However if more than 100 ul of chromatin is Fellet Frotein a Magnetic beads In each IF by placing the tubes in a Magnetic Sate Up 10 900 Hof Iysateiper 1 9 mi microcentrituga tubie Wiin several required per IP the cross linked chromatin preparation does not need to be diluted Separation Rack Wait 1 2 min for solution to clear and then carefully pulses to break nuclear membrane Incubate samples for 30 sec on wet ice q p j aa p p tant between pulses Optimal conditions required for complete lysis of nuclei can be as described below Antibodies can be added directly to the undiluted chromatin remove supernatant determined by observing nuclei on a light microscope before an
40. ls Place the microwells in the strip holder Unused microwells must be resealed in the storage bag and stored at 4 C immediately 2 Cell lysates can be used undiluted or diluted with sample diluent supplied in each PathScan Sandwich ELISA Kit blue color Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results 3 Add 50 ul of each undiluted or diluted cell lysate to the appropriate well Seal with tape and press firmly onto top of microwells Incubate the plate for 2 hr at room temperature Alternatively the plate can be incubated overnight at 4 C 4 Gently remove the tape and wash wells a Discard plate contents into a receptacle b Wash 4 times with 1X Wash Buffer 150 ul each time per well c For each wash strike plates on fresh paper towels hard enough to remove the residual solution in each well but do not allow wells to dry completely at any time d Clean the underside of all wells with a lint free tissue 5 Add 50 ul of detection antibody green color to each well Seal with tape and incubate the plate at room temperature for 1 hr 6 Repeat wash procedure Section C Step 4 7 Add 50 ul of HRP linked secondary antibody red color to each well Seal with tape and incubate the plate at room temperature for 30 min 8 Repeat wash procedure Section C Step 4 9 Prepare detection reagent working solution by mixing equal parts
41. lsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan Chromatin Immunoprecipitation ChIP lest updated 8 21 13 Troubleshooting Guide pg 1 of 2 a A Expected Chromatin Yield When harvesting cross linked chromatin from tissue samples the yield of chromatin can vary significantly between tissue types The table below provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 10 HeLa cells and the expected DNA concentration as determined in Section IV of the protocol For each tissue type disaggregation using a BD Medimachine system BD Biosciences or a Dounce homogenizer yielded similar amounts of chromatin However chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer A Dounce homogenizer is strongly recommended for disaggregation of brain tissue as the Medimachine does not adequately disaggregate brain tissue into a single cell suspension For optimal ChIP results we recommend using 5 to 10 bg of digested cross linked chromatin per IP therefore some tissues may require harvesting more than 25 mg per each IP To each of the 5 tubes in Step 2 add 0 pl 2 5 ul 5 pl 7 5 ul or 10 ul of the diluted micrococcal nuclease mix by inverting ube several times and incubate for 20 min at 37 C with frequent mixing S
42. mix and incubate 10 min at room temperature Final formaldehyde concentration is 1 Addition of formaldehyde may result in a color change of the medium 2 Add 2 ml of 10X glycine solution to each 15 cm dish containing 20 ml medium swirl briefly to mix and incubate 5 min at room temperature Addition of glycine may result in a color change of the medium For suspension cells transfer cells to a 50 ml conical tube centrifuge at 1 500 rpm in a bench top centrifuge for 5 min at 4 C and wash pellet two times with 20 ml ice cold 1X PBS Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion Section Ill 4 For adherent cells remove media and wash cells two times with 20 ml ice cold 1X PBS completely removing wash from culture dish each time 5 Add 2 ml ice cold 1X PBS PIC to each 15 cm dish Scrape cells into cold buf fer Combine cells from all culture dishes into one 15 ml conical tube 6 Centrifuge cells at 1 500 rpm in a bench top centrifuge for 5 min at 4 C Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion Section Ill co Ill Nuclei Preparation and Chromatin Digestion One IP preparation is defined as 25 mg of disaggregated tissue or 4 x 10 tissue culture cells Before starting e Remove and warm 200X Protease Inhibitor Cocktail PIC and 1 M DTT Make sure both are completely thawed and DTT crystals are completely in soluti
43. mmunoblotting or kinase activity Section D www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan Using Immobilized Antibodies Magnetic Bead Conjugate 1 Vortex gently and add immobilized bead conjugate 10 ul to 200 ul cell lysate at 1 mg ml Incubate with gentle rocking overnight at 4 C 2 Pellet magnetic beads by placing the tubes in a magnetic separation rack 7017 and wait 1 to 2 min for solution to clear Wash pellet five times with 500 ul of 1X cell lysis buffer Keep on ice during washes 3 Proceed to analyze by western immunoblotting or kinase activity Section D D Sample Analysis Proceed to one of the following specific set of steps NOTE For magnetic beads do not centrifuge Instead use a magnetic separation rack 7017 For Analysis by Western Immunoblotting 1 Resuspend the pellet with 20 HI 3X SDS sample buffer Vortex then microcentri fuge for 30 sec 2 Heat the sample to 95 100 C for 2 5 min and microcentrifuge for 1 min at 14 000 x g 3 Load the sample 15 30 pl on a 4 20 gel for SDS PAGE 4 Analyze sample by western blot see Western Immunoblotting Protocol NOTE For proteins with molecular weights near 50 kDa we recommend using Mouse Anti rabbit IgG Light Chain Specific L57A3 mAb 3677 or Mouse Anti rabbit IgG Conformation Specific L27A9 mAb 3678 as a secondary antibody to minimize masking produced by denatured
44. n 20 is a registered trademark of ICI Americas Inc A Cell Signaling TECHNOLOGY This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblotting or kinase activity A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L of 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 2 10X Cell Lysis Buffer 9803 To prepare 10 ml of 1X cell lysis buffer add 100 ml cell lysis buffer to 9 ml dH O mix NOTE Add 1 mM PMSF 8553 immediately prior to use 3 3X SDS Sample Buffer Blue Loading Pack 7722 or Red Loading Pack 7723 Prepare fresh 3X reducing loading buffer by adding 1 10 volume 30X DTT to 1 volume of 3X SDS loading buffer 4 Protein A or G Agarose Beads For unconjugated primary antibodies Use Protein A 9863 8687 for rabbit IgG immunoprecipitation and Protein G 8740 for mouse IgG immunoprecipitation NOTE Magnetic beads 8687 8740 require 6 Tube Magnetic Separation Rack 7017 5 Immobilized Streptavidin Bead Conjugate For biotinylated antibodies 3419 Gently vortex vial and use 10 pl per immunoprecipitation 6 10X Kinase Buffer for kinase assays 9802 To Prepare 1 ml of 1X kinase buffer add 100 ul 10X kinase buffer to 900 ul dH20 mix 7 ATP 10 mM for kinase assays 9804 To prepare 0 5 ml of ATP 200 uM add 10 pl
45. nd wet transfer for 1 5 hr at 70 V not a 0 45 um pore size membrane or transferring overnight which may result in over transfer and a lack of small protein signal Blocking Blocking the membrane for too long can obscure antigenic epitopes and prevent the antibody from binding Block for only 1 hr at room temperature Washing Washing for longer than the recommended three times 5 min is a common issue and can result in reduced signal We recommend that washes be timed to ensure accuracy TBST should contain 0 1 Tween 20 This applies to washing steps after both primary and secondary antibody incubations Additionally we recommend washing in TBST as washing in PBST has been shown to result in reduced signal Secondary antibody Serial dilutions of the secondary antibody can be performed on blots with the same cell extracts and primary antibody to optimize secondary antibody concentration Always incubate the secondary antibody in 5 nonfat dry milk in dilution and or incubation TBST for 1 hr at room temperature Avoid including sodium azide in HRP conjugated secondary antibody buffers as azide inhibits HRP activity CST secondary antibodies have already been optimized and do not require titration Detection reagent Use biotinylated molecular weight standards that can be detected with anti biotin HRP as positive controls for chemiluminescent detection 08 2013 Cell Signaling Technology Inc CST is a registered trademark of Cell Signaling Technology Twee
46. nicated and clarified tissue extracts may lessen background Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors for phospho targets Phosphatase Inhibitor Cocktail 100X 5870 Protease Phosphatase Inhibitor Cocktail 100X 5872 Protease Inhibitor Cocktail 5871 We recommend the use of Cell Lysis Buffer 9803 or RIPA Buffer 9806 which contains stronger detergent for sample preparation and protein concentration quantification SDS loading buffer 7722 or 7723 may be used for whole cell lysis without protein quantification Chaps Cell Extract Buffer 9852 is used to prepare cytoplasmic cell fractions and is useful for studying caspase signaling Cell Fractionation Kit 9038 is used to prepare cytoplasmic membrane organelle and nuclear cytoskeletal cell fractions Membrane Use high quality nitrocellulose membrane Nitrocellulose Sandwiches 12369 Pore size 0 2 um is generally recommended membranes with a pore size of 0 45 um are not recommended for proteins smaller than 30 kDa PVDF membranes may yield higher background than nitrocellulose Nylon membranes are not recommended for western blotting Block for 1 hr at room temperature in 5 nonfat dry milk in TBST Primary antibody dilution Incubate primary antibody overnight at 4 C in TBST at the recommended dilution with the recommended blocking agent either 5 BSA or 5 nonfat dry milk For individual antibodies please consult
47. o immunostaining Section D or store cells in PBS with 0 1 sodium azide at 4 C for intracellular staining proceed to permeabilization Section C C Permeabilization NOTE This step is critical for many CST antibodies 1 Permeabilize cells by adding ice cold 100 methanol slowly to pre chilled cells while gently vortexing to a final concentration of 90 methanol Alternatively remove fix prior to permeabilization by centrifugation and resuspension in 90 methanol 2 Incubate 30 min on ice 3 Proceed with immunostaining Section D or store cells at 20 C in 90 methanol 08 2013 Cell Signaling Technology Inc D Immunostaining NOTE Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies Count Cells using a hemocytometer or alternative method 1 Aliquot 0 5 1x108 cells into each assay tube by volume 2 Add 2 3 ml incubation buffer to each tube and rinse by centrifugation Repeat 3 Resuspend cells in 100 ul incubation buffer per assay tube 4 Block in Incubation Buffer for 10 min at room temperature 5 Add the unconjugated biotinylated or fluorochrome conjugated primary antibody at the appropriate dilution to the assay tubes see individual antibody datasheet or product webpage for the appropriate dilution 6 Incubate for 1 hr at room temperature 7 Wash by centrifugation in 2 3 ml incubation buffer 8 If using a fluorochrome conjugated primary antibo
48. o the following criteria Primer length 24 nucleotides Optimum Tm 60 C Optimum GC 50 Amplicon size 150 200 bp for standard PCR 80 160 bp for real time quantitative PCR Standard PCR Method 1 Label the appropriate number of 0 2 ml PCR tubes for the number of samples to be analyzed These should include the 2 input sample the positive control histone H3 sample the negative control normal rabbit IgG sample and a tube with no DNA to control for DNA contamination 2 Add 2 ul of the appropriate DNA sample to each tube 3 Prepare a master reaction mix as described below making sure to add enough reagent for two extra tubes to account for loss of volume Add 18 ul of master mix to each reaction tube Reagent Volume for 1 PCR Reaction 18 ul Nuclease free H O 12 5 pl 10X PCR Buffer 2 0 ul 4 mM dNTP Mix 1 0 pl 1 5 uM RPL30 Primers 2 0 pl Taq DNA Polymerase 0 5 pl 4 Start the following PCR reaction program a Initial denaturation 95 C 5 min b Denature 95 C 30 sec c Anneal 62 C 30 sec d Extension 72 C 30 sec e Repeat Steps b d for a total of 34 cycles f Final Extension 72 C 5 min 5 Remove 10 ul of each PCR product for analysis by 2 agarose gel or 10 poly acrylamide gel electrophoresis with a 100 bp DNA marker The expected size of the PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30 Real Time Quantitative PCR Method 1 Label the appropriate number of PCR tubes or PCR
49. om cn support China www cstj co jp support Japan PathScan Sandwich ELISA ELISA Colorimetric last updated 8 21 13 NOTE Refer to product specific datasheets or product webpage for assay incubation temperature A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare L PBS add 50 ml 10X PBS to 950 ml dH 0 mix 2 Bring all microwell strips to room temperature before use 3 Prepare 1X Wash Buffer by diluting 20X Wash Buffer included in each PathScan Sandwich ELISA Kit in dH 0 4 1X Cell Lysis Buffer 10X Cell Lysis Buffer 9803 To prepare 10 ml of 1X Cell Lysis Buffer add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH 0 mix PathScan Sandwich ELISA Lysis Buffer 7018 1X This buffer is ready to use as is Both buf fers can be stored at 4 C for short term use 1 2 weeks Recommended Add 1 mM phenylmethylsulfony fluoride PMSF 8553 immediately before use NOTE Refer to product specific datasheet or webpage for lysis buffer recommendation 5 TMB Substrate 7004 6 STOP Solution 7002 B Preparing Cell Lysates For adherent cells 1 Aspirate media when the culture reaches 80 90 confluence Treat cells by adding fresh media containing regulator for desired time 2 Remove media and rinse cells once with ice cold 1X PBS 3 Remove PBS and add 0 5 ml ice cold 1X cell lysi
50. on ia Cell Signaling TECHNOLOGY Chromatin Immunoprecipitation ChIP last updated 8 21 13 Enzymatic pg 2 of 3 Remove and warm 10X ChIP buffer and ensure SDS is completely in solution IV Analysis of Chromatin Digestion and Concentration 1 In one tube prepare enough 1X ChIP buffer for the dilution of digested chromatin e Prepare 1 ml 1X buffer A 250 pl 4X buffer A 750 ul water 0 5 pl 1 M DTT Recommended Step a into the desired number of IPs 400 l of 1X ChIP buffer 40 pl of 10X ChIP buffer 5 pl 200X PIC per IP prep and place on ice 1 To the 50 ul chromatin sample Step 9 in Section Ill add 100 hl nuclease free 360 ul H20 2 ul 200X PIC per IP When determining the number of IPs water 6 ul 5 M NaCl and 2 ul RNAse A Vortex to mix and incubate samples at remember to include the positive control Histone H3 D2B12 XP Rabbit mAb P 1 1 ml 1X Buffer B 275 ul 4X buffer B 825 pl wat 0 55 pl 1 M DIT 3 he an d ge besa 4A puter Bee Pale H 37 C for 30 min and negative control Normal Rabbit IgG antibody samples Place mix on ice e Prepare 100 pI 1X ChIP buffer 10 pI 10X ChIP Buffer 90 pl water 0 5 pl 2 To each RNAse A oigasted sample add 2 p Proteinase K vortex to mixand 2 To the prepared 1X ChIP buffer add the equivalent of 100 pl 5 10 pg of 200X PIC per IP prep and place on ice incubate samples at 65 C for 2 hr chromatin of the digested
51. or 2 hr Brain 2 5 pg per 25 mg tissue 20 50 pg ml 13 Remove 20 ul of each sample and determine DNA fragment size by electrophoresis on a 1 agarose gel with a 100 bp DNA Heart 2 5 ug per 25 mg tissue 20 50 ug ml marker 14 Observe which of the digestion conditions produces DNA in the desired range of 150 900 base pairs 1 6 nucleosomes ea 6 HeLa 10 15 pg per 4 x 10 cells 100 150 pg ml The volume of diluted micrococcal nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of micrococcal nuclease stock that should be added to one IP preparation 25 mg of disaggregated tissue cells or 4 X 108 tissue culture cells to produce the desired size of DNA fragments For example if 5 pl B Optimization of Chromatin Digestion Optimal conditions for the digestion of cross linked chromatin DNA to 150 900 bp in length is highly dependent on the ratio of of diluted micrococcal nuclease produces DNA fragments of 150 900 bp in this protocol then 0 5 ul of stock micrococcal micrococcal nuclease to the amount of tissue or number of cells used in the digest Below is a protocol for determination of the nuclease should be added to one IP preparation during the digestion of chromatin in Section Ill optimal digestion conditions for a specific tissue or cell type 15 If results indicate that DNA is not in the desired size range then repeat optimization protocol adjusting the amount of 1 Pr
52. ould be stored at 80 C until further use Remove 50 ull of the e Prepare high salt wash 1 ml 1X ChIP buffer 100 pl 10X ChIP buffer 900 ul H20 Separation Rack Wait 1 2 min for solution to clear and then carefully remove chromatin preparation for Analysis of Chromatin Digestion and Concentration 70 ul 5M NaCl per IP Store at room temperature until use supernatant and proceed to Section VI Section IV www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan Chromatin Immunoprecipitation ChIP Enzymatic pg 3 of 3 last updated 8 21 13 VI Elution of Chromatin from Antibody Protein G Agarose Beads and Reversal of Cross links Before starting e Remove and warm 2X ChIP elution buffer in a 37 C water bath and ensure SDS is in solution e Set a water bath or thermomixer to 65 C e Prepare 150 pl 1X ChIP elution buffer 75 pl 2X ChIP elution buffer 75 ul water for each IP and the 2 input sample 1 Add 150 ul of the 1X ChIP elution buffer to the 2 input sample tube and set aside at room temperature until Step 6 2 Add 150 ul 1X ChIP elution buffer to each IP sample 3 Elute chromatin from the antibody Protein G Beads for 30 min at 65 C with gentle vortexing 1 200 rpm A thermomixer works best for this step Alterna tively elutions can be performed at room temperature with rotation but may not be as complete 4 a Pellet Protein G Agarose Beads by brief 1 m
53. re processing of more than 25 mg per IP The amount of tissue can be increased to 50 mg per IP while still maintaining efficient chromatin fragmentation and extraction Increase the number of sonications following chromatin digestion Visualize cell nuclei under micro scope before and after sonication to confirm complete lysis of nuclei Chromatin is under digested and fragments are too large greater than 900 bp Large chromatin fragments can lead to increased background and lower resolution Too many cells or not enough micrococcal nuclease was added to the chromatin digestion Tissue or cells may have been over cross linked Cross linking for longer than 10 min may inhibit digestion of chromatin Weigh tissue or count a separate plate of cells prior to cross linking to determine accurate cell number Add less tissue or cells or more micrococcal nuclease to the chromatin digest See Section B for optimization of chromatin digestion 5 Perform a time course at a fixed formaldehyde concentration Shorten the time of cross linking to 10 min or less Chromatin is over digested and fragments are too small exclusively 150 bp mono nucleosome length Complete digestion of chromatin to mono nucleosome length DNA may diminish signal during PCR quantification especially for amplicons greater than 150 bp in length Not enough cells or too much micrococcal nuclease added to the chromatin digestion Weigh tissue or count a separate
54. s Buffered Saline TBS 12498 To prepare 1 L 1X TBS add 100 ml 10X to 900 ml dH 0 mix 3 1X SDS Sample Buffer Blue Loading Pack 7722 or Red Loading Pack 7723 Prepare fresh 3X reducing loading buffer by adding 1 10 volume 30X DTT to 1 volume of 3X SDS loading budder Dilute to 1X with dH 0 4 10X Tris Glycine SDS Running Buffer 4050 To prepare 1 L 1X running buffer add 100 ml 10X running buffer to 900 ml dH 0 mix 10X Tris Glycine Transfer Buffer 12539 To prepare 1 L 1X Transfer Buffer add 100 ml 10X Transfer Buffer to 200 ml methanol 700 ml dH 0 mix 10X Tris Buffered Saline with Tween 20 TBST 9997 To prepare 1 L 1X TBST add 100 ml 10X TBST to 900 ml dH 0 mix Nonfat Dry Milk 9999 Blocking Buffer 1X TBST with 5 w v nonfat dry milk for 150 ml add 7 5 g nonfat dry milk to 150 ml 1X TBST and mix well 9 Wash Buffer 9997 1X TBST 10 Bovine Serum Albumin BSA 9998 11 Primary Antibody Dilution Buffer 1X TBST with 5 BSA or 5 nonfat dry milk as indicated on primary antibody datasheet for 20 ml add 1 0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well 12 Biotinylated Protein Ladder Detection Pack 7727 13 Prestained Protein Marker Broad Range Premixed Format 7720 14 Blotting Membrane and Paper 12369 This protocol has been optimized for nitrocellulose membranes Pore size 0 2 um is generally recommended 15 Secondary Antibody Conjugated to HRP anti rabb
55. s buffer plus 1 mM PMSF to each plate 10 cm diameter and incubate the plate on ice for 5 min 4 Scrape cells off the plate and transfer to an appropriate tube Keep on ice 5 Sonicate lysates on ice 6 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in single use aliquots For suspension cells 1 Remove media by low speed centrifugation 1 200 rpm when the culture reaches 0 5 1 0 x 10 viable cells ml Treat cells by adding fresh media containing regulator for desired time 2 Collect cells by low speed centrifugation 1 200 rpm and wash once with 5 10 ml ice cold 1X PBS 3 Cells harvested from 50 ml of growth media can be lysed in 2 0 ml of 1X cell lysis buffer plus 1 mM PMSF 4 Sonicate lysates on ice 5 Microcentrifuge for 10 min x14 000 rpm at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Store at 80 C in single use aliquots 08 2013 Cell Signaling Technology Inc PathScan is a registered trademark of Cell Signaling Technology C Test Procedure 1 After the microwell strips have reached room temperature break off the required number of microwells Place the microwells in the strip holder Unused microwells must be resealed in the storage bag and stored at 4 C immediately 2 Cell lysates can be undiluted or diluted with sample diluent supplied in each PathScan Sandwich
56. s in wash buffer for 5 min 5 Block each section with 100 400 ul blocking solution for 1 hr at room temperature 6 Remove blocking solution and add 100 400 yl primary antibody diluted in recommended antibody diluent to each section Incubate overnight at 4 C 7 Equilibrate SignalStain Boost Detection Reagent to room temperature 8 Remove antibody solution and wash sections with wash buffer three times for 5 min each 9 Cover section with 1 3 drops SignalStain Boost Detection Reagent as needed Incubate in a humidified chamber for 30 min at room temperature 10 Wash sections three times with wash buffer for 5 min each 11 Add 1 drop 30 pl SignalStain DAB Chromogen Concentrate to 1 ml SignalStain DAB Diluent and mix well before use 12 Apply 100 400 pl SignalStain DAB to each section and monitor closely 1 10 min generally provides an acceptable staining intensity 13 Immerse slides in dH 0 14 If desired counterstain sections with hematoxylin per manufacturer s instructions 15 Wash sections in dH O two times for 5 min each 16 Dehydrate sections a Incubate sections in 95 ethanol two times for 10 sec each b Repeat in 100 ethanol incubating sections two times for 10 sec each c Repeat in xylene incubating sections two times for 10 sec each 17 Mount sections with coverslips A Cell Signaling TECHNOLOGY Immunohistochemistry IHC Frozen using SignalStain Boost Detection Reagent las
57. s recommended in the product datasheet to 200 yl cell lysate at 1 mg ml Incubate with gentle rocking overnight at 4 C 2 Add either protein A or G agarose or magnetic beads 10 30 ul of 50 bead slurry Incubate with gentle rocking for 1 3 hr at 4 C for agarose beads or 10 30 min for magnetic beads 3 Microcentrifuge for 30 sec at 4 C Wash pellet five times with 500 ul of 1X cell lysis buffer Keep on ice between washes 4 Proceed to analyze by western immunoblotting or kinase activity Section D Using Biotinylated Primary Antibodies 1 Add biotinylated antibody at the appropriate dilution as recommended in the product datasheet to 200 pl cell lysate at 1 mg ml Incubate with gentle rocking overnight at 4 C 2 Gently mix Immobilized Streptavidin Sepharose Bead Conjugate 3419 and add 10 ul of slurry Incubate with gentle rocking for 2 hr at 4 C 3 Microcentrifuge for 30 sec at 4 C Wash pellet five times with 500 ul of 1X cell lysis buffer Keep on ice during washes 4 Proceed to analyze by western immunoblotting or kinase activity Section D Using Immobilized Antibodies Sepharose Bead Conjugate 1 Vortex gently and add immobilized bead conjugate 10 pl to 200 hl cell lysate at 1 mg ml Incubate with gentle rocking overnight at 4 C 2 Microcentrifuge for 30 sec at 4 C Wash pellet five times with 500 ul of 1X cell lysis buffer Keep on ice during washes 3 Proceed to analyze by western i
58. single use aliquots 08 2013 Cell Signaling Technology Inc PathScan is a registered trademark of Cell Signaling Technology Tween 20 is a registered trademark of ICI Americas Inc C Coating Procedure 1 Rinse microplate with 200 HI of dH 0 discard liquid Blot on paper towel to make sure wells are dry 2 Dilute capture antibody 1 100 in 1X PBS For a single 96 well plate add 100 hl of capture antibody stock to 9 9 ml 1X PBS Mix well and add 100 l well Cover plate and incubate overnight at 4 C 17 20 hr 3 After overnight coating gently uncover plate and wash wells a Discard plate contents into a receptacle b Wash four times with wash buffer 200 ul each time per well For each wash strike plates on fresh paper towels hard enough to remove the residual solution in each well but do not allow wells to completely dry at any time c Clean the underside of all wells with a lint free tissue 4 Block plates Add 150 ul of blocking buffer well cover plate and incubate at 37 C for 2 hr 5 After blocking wash plate Section C Step 3 Plate is ready to use D Test Procedure 1 Lysates can be used undiluted or diluted in blocking buffer 100 ul of lysate is added per well Cover plate and incubate at 37 C for 2 hr 2 Wash plate Section C Step 3 3 Dilute detection antibody 1 100 in blocking buffer For a single 96 well plate add 100 ul of detection antibody Stock to 9 9 ml of blocking buffer Mix well and add
59. t updated 8 21 13 IMPORTANT Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine whether a product is validated and approved for use frozen tissue sections Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution NOTE Please see product datasheet and website for product specific protocol recommendations A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 Xylene 2 Ethanol anhydrous denatured histological grade 100 and 95 3 Hematoxylin optional 4 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH20 mix 5 Fixative Options For optimal fixative please refer to the product datasheet a 10 neutral buffered formalin b Acetone c Methanol d 3 formaldehyde To prepare 100 ml add 18 75 ml 16 formaldehyde to 81 25 ml 1X PBS 6 10X Tris Buffered Saline TBS Wash Buffer 12498 To prepare 1 L 1X TBS add 100 ml of 10X TBS to 900 ml dH 0 mix 7 Methanol Peroxidase To prepare add 10 ml 30 H O to 90 ml methanol Store at 20 C 8 Blocking Solution 1X TBS 0 3 Triton X 100 5 Normal Goat Serum 5425 To prepare add 500 ul goat serum and 30 ul Triton X 100 to 9 5 ml 1X TBS 9 Detection System SignalStain Boost IHC Detection Reagents HRP Mouse 8125 HAP Rabbit 8114 10 Substrate SignalS
60. tain DAB Substrate Kit 8059 B Sectioning 1 For tissue stored at 80 C Remove from freezer and equilibrate at 20 C for approximately 15 min before attempting to section This may prevent cracking of the block when sectioning 2 Section tissue at a range of 6 8 bm and place on positively charged slides 3 Allow sections to air dry on bench for a few min before fixing this helps sections adhere to slides C Fixation Options NOTE Consult product datasheet to determine the optimal fixative 1 After sections have dried on the slide fix in optimal fixative as directed below a 10 Neutral buffered formalin 10 min at room temperature Proceed with staining procedure immediately Section D b Cold acetone 10 min at 20 C Air dry Proceed with staining immediately Section D c Methanol 10 min at 20 C Proceed with staining immediately Section D d 3 Formaldehyde 15 min at room temperature Proceed with staining immediately Section D e 3 Formaldehyde methanol 15 min at room temperature in 3 formaldehyde followed by 5 min in methanol at 20 C do not rinse in between Proceed with staining immediately Section D 08 2013 Cell Signaling Technology Inc SignalStain and CST are trademarks of Cell Signaling Technology Triton X 100 is a trademark of The Dow Chemical Company D Staining o 1 1 1 1 1 1 1 1 N Aa Ww N O PINIM PiN Wash sections in wash buff
61. te membrane in 25 ml of blocking buffer for 1 hr at room temperature 3 Wash three times for 5 min each with 15 ml of TBST Il Primary Antibody Incubation Proceed to one of the following specific set of steps depending on the primary antibody used For Unconjugated Primary Antibodies 1 Incubate membrane and primary antibody at the appropriate dilution and diluent as recommended in the product datasheet in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4 C Wash three times for 5 min each with 15 ml of TBST Incubate membrane with the species appropriate HRP conjugated secondary antibody 7074 or 7076 at 1 2000 and anti biotin HRP linked Antibody 7075 at 1 1000 1 3000 to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature Wash three times for 5 min each with 15 ml of TBST Proceed with detection Section D won aa 1 Incubate membrane and primary antibody at the appropriate dilution as recom mended in the product datasheet in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4 C Wash three times for 5 min each with 15 ml of TBST Incubate with Anti biotin HRP linked Antibody 7075 at 1 1000 1 3000 to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature 4 Wash three times for 5 min each with 15 ml of TBST 5 Pro
62. ted fixed and stained directly in multi well plates chamber slides or on coverslips 1 Aspirate liquid then cover cells to a depth of 2 3 mm with 4 formaldehyde diluted in warm PBS NOTE Formaldehyde is toxic use only in a fume hood 2 Allow cells to fix for 15 min at room temperature 3 Aspirate fixative rinse three times in 1X PBS for 5 min each 4 Proceed with Immunostaining Section NOTE Do not allow slides to dry at any time during this process 1 Deparaffinization Rehydration a Wash three times in xylene for 5 min each b Wash two times in 100 ethanol for 10 min each c Wash two times in 95 ethanol for 10 min each d Rinse sections two times in dH20 for 5 min each 2 Antigen Unmasking NOTE Consult product datasheet or product webpage for specific recommendation for the unmasking solution a For Citrate Bring slides to a boil in 10 mM sodium citrate buffer pH 6 0 then maintain at a sub boiling temperature for 10 min Cool slides on bench top for 30 min b For EDTA Bring slides to a boil in 1 mM EDTA pH 8 0 followed by 15 min at a sub boiling temperature No cooling is necessary 3 Proceed with Immunostaining Section C 1 For fixed frozen tissue proceed with Immunostaining Section C 2 For fresh unfixed frozen tissue fix immediately as follows a Cover sections with 4 formaldehyde diluted in warm 1X PBS b Allow sections to fix for 15 min at room temperature c Rinse slides three
63. ted mouse on mouse staining Include a control slide stained without the primary antibody to confirm whether the secondary antibody is the source of the background Washes Adequate washing is critical for contrasting low background and high signal Wash slides three times for 5 min with TBST 9997 after primary and secondary antibody incubations 08 2013 Cell Signaling Technology Inc CST is a trademark of Cell Signaling Technology A Cell Signaling TECHNOLOGY Notes www cellsignal com support USA amp Europe www cst c com cn support China www cstj co jp support Japan A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 2 16 Formaldehyde methanol free 3 100 methanol 4 Incubation Buffer Dissolve 0 5 g Bovine Serum Albumin BSA 9998 in 100 ml 1X PBS Store at 4 C 5 Secondary Antibodies Anti mouse 4408 8890 4410 8887 Anti rabbit 4412 8889 4414 8885 Anti rat 4416 4418 B Fixation 1 Collect cells by centrifugation and aspirate supernatant 2 Resuspend cells briefly in 0 5 1 ml 1X PBS Add formaldehyde to a final concentration of 4 formaldehyde 3 Fix for 10 min at 37 C 4 Chill tubes on ice for 1 min 5 For extracellular staining with antibodies that do not require permeabilization proceed t
64. the collection tube 5 Transfer remaining 375 ul of each sample from Step 1 to the spin column in collection tube Repeat Steps 3 and 4 6 Add 700 ul of DNA wash reagent B to the spin column in collection tube 7 Centrifuge at 14 000 rpm in a microcentrifuge for 30 sec 8 Remove spin column from the collection tube and discard the liquid Replace spin column in the collection tube 9 Centrifuge at 14 000 rpm in a microcentrifuge for 30 sec 10 Discard collection tube and liquid Retain spin column 11 Add 50 ul of DNA elution reagent C to each spin column and place into a clean 1 5 ml microcentrifuge tube 12 Centrifuge at 14 000 rpm in a microcentrifuge for 30 sec to elute DNA 13 Remove and discard DNA purification spin column Eluate is now purified DNA Samples can be stored at 20 C VIII Quantification of DNA by PCR Recommendations e Use Filter tip pipette tips to minimize risk of contamination e The control primers included in the kit are specific for the human or mouse RPL30 gene and can be used for either standard PCR or quantitative real time PCR If the user is performing ChiPs from another species it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions e A Hot Start Taq polymerase is recommended to minimize the risk of non specific PCR products e PCR primer selection is critical Primers should be designed with close adherence t
65. the product datasheet for recom and or incubation mended dilution buffer Inadequate washing Washing for less time than the recommended three times 5 min in TBST is common and can result in high background We recommend that washes be timed to ensure accuracy Additionally low Tween 20 can contribute to high background we recommend 0 1 Tween 20 This applies to washing steps after both primary and secondary antibody incubations Secondary antibody Some secondary antibodies bind nonspecifically to proteins in cell extracts To assess the quality of a secondary antibody perform a blot through to film exposure without primary antibody Serial dilutions of the secondary antibody dilution and or incubation can be performed on blots with the same cell extracts and primary antibody to optimize secondary antibody concentration Always incubate the secondary antibody in 5 nonfat dry milk in TBST for 1 hr at room temperature diluting the secondary antibody in BSA yields higher levels of background bands CST secondary antibodies have already been optimized and do not require titration Detection reagent Chemiluminescent detection requires that high purity water be used to dilute concentrated reagents Use only purified water with organic and inorganic impurities removed We recommend using RODI water Additionally ensure that the membrane is never allowed to dry out during the antibody incubation process prior to detection reagent exposure Exposure
66. top each digest by adding 10 pl of 0 5 M EDTA and placing tubes on ice Pellet nuclei by centrifugation at 13 000 rpm in a microcentrifuge for 1 min at 4 C and remove supernatant Resuspend nuclear pellet in 200 ul of 1X ChIP buffer PIC Incubate on ice for 10 min Sonicate lysate with several pulses to break nuclear membrane Incubate samples for 30 sec on wet ice between pulses Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei on a light microscope before and after sonication HeLa nuclei were completely lysed after 3 sets of 20 sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer Sonicator set at setting 6 with a 1 8 inch probe Alternatively nuclei can be lysed by homogenizing the lysate 20 imes in a Dounce homogenizer however lysis may not be as complete on oO ao Tissue Cell Total Chromatin Yield Expected DNA Concentration 9 Clarify lysates by centrifugation at 10 000 rpm in a microcentrifuge for 10 min at 4 C Spleen 20 30 ug per 25 mg tissue 200 300 ug ml 10 Transfer 50 ul of each of the sonicated lysates to new microfuge tubes Liver 10 15 pg per 25 mg tissue 100 150 ug ml 11 To each 50 pI sample add 100 ul nuclease free water 6H 5 M NaCl and 2 ul RNAse A Vortex to mix and incubate samples at 37 C for 30 min Kidney 8 10 pg per 25 mg tissue 80 100 pg ml 12 To each RNAse A digested sample add 2 pl Proteinase K Vortex to mix and incubate sample at 65 C f
67. ube and centrifuge at 1 500 rpm in a bench top centrifuge for 5 min at 4 C 4 Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion Section Ill 08 2013 Cell Signaling Technology Inc SimpleChIP and XP are registered trademarks of Cell Signaling Technology Il Cell Culture Cross linking and Sample Preparation For optimal ChIP results use approximately 4 x 10 cells for each IP to be performed For HeLa cells this is equivalent to half of a 15 cm culture dish containing cells that are 90 confluent in 20 ml of growth medium One additional sample should be processed for Analysis of Chromatin Digestion and Concentration Section IV Include one extra dish of cells in experiment to be used for determination of cell mber using a hemocytometer Before starting Remove and warm 200X Protease Inhibitor Cocktail PIC and 10X Glycine Solution Make sure PIC is completely thawed Prepare 2 ml of Phosphate Buffered Saline PBS 10 ul 200X PIC per 15 cm dish to be processed and place on ice Prepare 40 ml of 1X PBS per 15 cm dish to be processed and place on ice Prepare 540 ul of 37 formaldehyde per 15 cm dish of cells to be processed and eep at room temperature Use fresh formaldehyde that is not past the manufac urer s expiration date 1 To crosslink proteins to DNA add 540 ul of 37 formaldehyde to each 15 cm culture dish containing 20 ml medium Swirl briefly to
68. ver the world s highest quality research diagnostic and therapeutic products that accelerate biological understanding and enable personalized medicine ai Lil FOUNDED BY RESEARCH SCIENTISTS IN 1999 Cell Signaling Technology CST is a private family owned company with over 400 employees worldwide Active in the field of applied systems biology research particularly as it relates to cancer CST understands the importance of using antibodies with high levels of specificity and lot to lot consistency It s why we produce all of our antibodies in house and perform painstaking validations for multiple applications And the same CST scientists who produce our antibodies also provide technical support for customers helping them design experiments troubleshoot and achieve reliable results We do this because that s what we d want if we were in the lab Because actually we are D Immunohistochemistry Group Danvers MA USA p O O a aaa a Cell 11 Signaling TECHNOLOGY ial Cell Signaling TECHNOLOGY UNITED STATES CHINA Hours M F 8 30am to 8 00em EST Tel 86 21 58356288 Orders 877 616 2355 orders cellsignal com Support China 4006 473287 GreatQ tech cst c com cn Support 877 678 8324 support cellsignal com Support Asia Pacific csttechasia cst c com cn www cellsignal com www cst c com cn WWW CELLSIGNAL COM Printed in the USA on recycled paper 25 post consumer waste fiber using vegetable in
69. ww cst c com cn support China www cstj co jp support Japan last updated 8 21 13 Immunohistochemistry IHC Troubleshooting Guide Suboptimal IHC staining is frequently resolved by adjusting relatively few variables Adjustments to key steps within the protocol such as antigen retrieval can often resolve these common issues LITTLE OR NO STAINING Sample storage Slides may lose signal over time in storage This process is variable and dependent upon the protein target The effect of slide storage on staining has not been established for every protein therefore it is best practice that slides are freshly cut before use If slides must be stored do so at 4 C Do not bake slides before storage Tissue sections dried out It is vital that the tissue sections remain covered in liquid throughout the staining procedure Slide preparation Inadequate deparaffinization may cause spotty uneven background staining Repeat the experiment with new sections using fresh xylene Antigen unmasking retrieval Fixed tissue sections have chemical crosslinks between proteins that dependent on the tissue and antigen target may prevent antibody access or mask antigen targets Antigen unmasking protocols may utilize a hot water bath microwave or pressure cooker Antigen unmasking protocols utilizing a water bath are not recommended Antigen unmasking performed with a microwave is preferred though staining of particular tissues or antigen targets may requir
70. y LumiGLO is a registered trademark of Kirkaard and Perry Laboratories Tween 20 is a registered trademark of ICI Americas Inc ia Cell Signaling TECHNOLOGY For western blots incubate membrane with diluted primary antibody in either 5 w v BSA or nonfat dry milk 1X TBS 0 1 Tween 20 at 4 C with gentle shaking overnight NOTE Two color western blots require primary antibodies from different species and ap propriate secondary antibodies labeled with different dyes Overlap of epitopes may cause interference and should be considered in two color western blots If the primary antibodies require different primary antibody incubation buffers test each primary individually in both buffers to determine the optimal one for the dual labeling experiment A Solutions and Reagents NOTE Prepare solutions with reverse osmosis deionized RODI or equivalent grade water 1 1 1 1 1 1 20X Phosphate Buffered Saline PBS 9808 To prepare 1 L 1X PBS add 50 ml 20X PBS to 950 ml dH 0 mix 2 10X Tris Buffered Saline TBS 12498 To prepare 1 L 1X TBS add 100 ml 10X TBS to 900 ml dH 0 mix 3 1X SDS Sample Buffer Blue Loading Pack 7722 or Red Loading Pack 7723 Prepare fresh 3X reducing loading buffer by adding 1 10 volume 30X DTT to 1 volume of 3X SDS loading budder Dilute to 1X with dH 0 4 10X Tris Glycine SDS Running Buffer 4050 To prepare 1 L 1X running buffer add 100 ml 10X running buffer 900 ml dH 0 mix 5

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CST Protocols & Troubleshooting Guides