SANTA CRUZ BIOTECHNOLOGY, INC. PROTOCOLS 1.WESTERN (IMMUNO-)BLOTTING

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1 SANTA CRUZ BIOTECHNOLOGY, INC. PROTOCOLS 1.WESTERN (IMMUNO-)BLOTTING A. Sample Preparation NOTE: For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents. MONOLAYER CELLS Grow cells to subconfluency in a 100 mm x 20 mm petri dish, remove culture medium and rinse cell monolayer with room temperature 1x PBS (10X liquid PBS: sc-24946). The following steps should be performed on ice or at 4 C using fresh, ice cold buffers. Add 0.6 ml of RIPA buffer (sc-24948) to the monolayer cells in the plate. Gently rock plate for 15 minutes at 4 C. Remove adherent cells with a cell scraper. Transfer the resulting lysate to a microcentrifuge tube. Wash the plate once with 0.3 ml of RIPA buffer and combine with first lysate. (Optional: Add 10 µl of 10 mg/ml PMSF (cat # sc-3597) stock and/or pass through a 21-gauge needle to shear the DNA.) Incubate minutes on ice. Centrifuge cell lysate at 10,000xg for 10 minutes at 4 C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microcentrifuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants. NOTE: For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology Inc. s PhosphoCruz Protein Purification System (sc ). SUSPENSION CELLS Collect approximately 2.0 x 10 7 cells by low-speed centrifugation (e.g. 200xg) at room temperature for 5 minutes. Carefully remove culture medium. Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation. Carefully remove supernatant. Add 1.0 ml of ice cold RIPA buffer (sc-24948) with freshly added protease inhibitors and/or phosphatase inhibitors. Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minutes. Further disrupt and homogenize cells by hydrodynamic shearing (21-gauge needle), dounce homogenization or sonication, taking care not to raise the temperature of the lysate. (Optional: Add 10 µl of 10 mg/ml PMSF stock; sc-3597) Incubate 30 minutes on ice. Transfer to microcentrifuge tube(s) and centrifuge at 10,000xg for 10 minutes at 4 C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microcentrifuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants. NOTE: For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology Inc. s PhosphoCruz Protein Purification System (sc-24964). TISSUE SAMPLES Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue should be sliced very thinly and thawed in RIPA buffer (sc ) containing protease inhibitors and/or phosphatase inhibitors. Use 3 ml of ice cold RIPA buffer per gram of tissue. Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, maintaining temperature at 4 C throughout all procedures. (Optional: Add 30 µl of 10 mg/ml PMSF (sc-3597) stock per gram of tissue.) Incubate on ice for 30 minutes. Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4 C. Remove supernatant and centrifuge it again. The supernatant fluid is the total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate. NOTE: For phosphorylation studies, lysates may be enriched for phosphoproteins using Santa Cruz Biotechnology, Inc. s PhosphoCruz Protein Purification System (sc-24964). B. Electrophoresis Mix sample (40 60 µg whole cell lysate, µg nuclear extract or ng purified protein per lane) with an equal volume of 2x electrophoresis sample buffer (sc-24945) and boil for 2 3 minutes. Unused samples may be stored at -20 C. Load up to 10 µl of lysate per 1.0 mm of well width for gels of 0.75 mm thickness. We recommend the use of Cruz Marker molecular weight standards (sc-2035). Load 2 µl/well for 0.75 mm gels and 5 µl/well for 1.5 mm gels. When used with Cruz Marker -compatible secondary antibodies, internal standard bands will appear when the probed blot is exposed to detection reagent. Alternatively, use Prestained Molecular Weight Standards (sc-2361). Electrophorese according to standard protocols. Transfer proteins from the gel to a nitrocellulose or PVDF membrane using an electroblotting apparatus according to the manufacturer s protocols. NOTE: Ready-made blots of human or mouse whole cell extracts or nuclear extracts or mouse tissues are available as Cruz Blot Systems. 1

2 C. Immunoblotting Block non-specific binding by incubating membrane in Blotto (either Blotto A or Blotto B; IgG-free BSA, sc-2323, is recommended when using antibovine secondary antibodies) for minutes at room temperature. Alternatively, the membrane may be blocked at 4 C overnight in a covered container, using Blotto without Tween-20. If using a phospho-specific antibody, add 0.01% (v/v) of each Phosphatase Inhibitor Cocktails A and B (sc and sc-45045) to the blocking solution and the antibody diluent to inhibit phosphatases. Incubate the blocked membrane in primary antibody diluted in Blotto for 1 hour at room temperature. (For phospho-specific antibodies: Use Blotto with 0.01% (v/v) of each Phosphatase Inhibitor Cocktails A and B (sc and sc ) Optimal antibody concentration should be determined by titration. We recommend a starting dilution of µg/ml. Wash membrane three times for 5 minutes each with TBST. Incubate the membrane for 45 minutes at room temperature with horseradish peroxidase (HRP) conjugated secondary antibody, or alkaline phosphatase (AP) conjugated secondary antibody, diluted to 1:500 1:2000 in Blotto. If high backgrounds are observed, secondary antibody should be diluted further (up to 1:20,000). If Cruz Marker molecular weight standards (sc-2035) are used in the gel, the Cruz Marker compatible secondary antibodies must be used in order to visualize standards with ECL. Wash membrane three times for 5 minutes each with TBST and once for 5 minutes with TBS. Incubate membrane in Chemiluminescence Luminol Reagent (sc-2048) according to Luminol data sheet, or visualize proteins using standard protocols. If luminol is used for visualization, an HRP-conjugated secondary antibody must be used. 2.IMMUNOPRECIPITATION NOTE: This procedure may be used for cells labeled with radioactive compounds such as amino acids or orthophosphate. (Radioisotope use and disposal should conform to institutional and governmental regulations.) Cell labeling should be carried out in medium lacking the relevant nonradioactive compound. Starving cells appropriately prior to labeling is recommended. Incubate cultured cells (80 90% confluent monolayer in 100 mm cell culture plate or approximately 2 5 x 10 7 suspension cells in flask). Example: Following starvation, remove culture medium and replace with methionine-free medium containing 5% dialyzed fetal calf serum and 100 µci/ml 35 S-methionine. Incubate 1 hour at 37 C. For some proteins a longer labeling period (up to 18 hours) is preferable. Wash cells with PBS as necessary to remove unincorporated 35 S-methionine. NOTE: For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents. Add 1 3 ml ice cold RIPA buffer (sc-24948) to subconfluent cell monolayer and incubate at 4 C for 10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a microcentrifuge tube. Disrupt cells by repeated passage through a 21-gauge needle or sonication. Transfer to a microcentrifuge tube. Optional: Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer and combine with original extract. Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4 C. Transfer supernatant to a fresh microcentrifuge tube on ice. Preclear lysate by adding 1.0 µg of the appropriate control IgG (corresponding to the host species of the primary antibody), together with 20 µl of appropriate suspended (25% v/v) agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Incubate at 4 C for 30 minutes. Centrifuge at 3,000 rpm (e.g. Eppendorf 5415D; approximately 1,000xg) for 30 seconds at 4 C. Transfer the supernatant, or approximately µg total cellular protein, to a microcentrifuge tube. Add 1 10 µl (i.e., µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 2 hours at 4 C. Alternatively, if antibody agarose conjugate is employed, add 20 µl (i.e., 5 µl packed beads) and incubate at 4 C for 1 hour to overnight with mixing; skip the next step. Add 20 µl of the appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Cap tubes and incubate at 4 C on a rocker platform or rotating device for 1 hour to overnight. Collect immunoprecipitates by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4 C. Carefully aspirate and discard supernatant. Gently wash pellet 2 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above. After final wash, carefully aspirate and discard supernatant and resuspend pellet in 40 µl of 2x electrophoresis sample buffer (sc-24945). Boil samples for 2 3 minutes and subject to electrophoresis and autoradiography. Unused samples may be stored at -20 C. Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant. 2

3 3. IMMUNOPRECIPITATION/WESTERN BLOTS NOTE: For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents. Prepare a total cell lysate as described under Western blot procedure on page 1. NOTE: For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology, Inc. s PhosphoCruz Protein Purification System (sc-24964). Preclear whole cell lysate (optional step) as follows. To approximately 1 ml of whole cell lysate or tissue extract, add 0.25 µg of the appropriate control IgG (corresponding to the host species of the primary antibody), together with 20 µl of appropriate suspended (25% v/v) agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose, see table below). Incubate at 4 C for 30 minutes. Pellet beads by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4 C. Transfer supernatant (cell lysate) to a new microcentrifuge tube at 4 C. To 1 ml of the above cell lysate, or approximately µg of total cellular protein, add 10 µg of primary antibody agarose conjugate (i.e., 5 µl volume of packed beads) and incubate at 4 C for 1 hour to overnight with mixing. Alternatively, if primary antibody agarose conjugate is not available, incubate 1 ml cell lysate with 1 10 µl (i.e., µg) primary antibody (optimal antibody concentration should be determined by titration) for 1 2 hours at 4 C. Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose or Protein L-Agarose). Cap tubes and incubate at 4 C on a rocker platform or rotating device for 1 hour to overnight. Collect pellet by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4 C. Carefully aspirate and discard supernatant. Wash pellet 2 4 times with either RIPA buffer (sc-24948) (more stringent) or PBS (less stringent), each time repeating centrifugation step above. After final wash, aspirate and discard supernatant and resuspend pellet in 40 µl of 2x electrophoresis sample buffer (sc-24945). Boil samples for 2 3 minutes. Load up to 5 10 µl of sample per 1.0 mm well width for gels of 0.75 mm thickness. Continue with electrophoresis and immunoblotting as described under Western blotting procedure on page 1. NOTE: Depending on the secondary antibody that is used, 55 kda and 27 kda heavy and light IgG chains, respectively, of the primary antibody may be detected. These bands will be less pronounced if a primary antibody agarose conjugate is used in the above procedure or if ExactaCruz Reagents are used. 4. EXACTACRUZ IMMUNOPRECIPITATION/WESTERN BLOTS Prepare a total cell lysate as described under Western (Immuno-) blotting procedure. Preclear whole cell lysate (optional): To approximately 1 ml of whole cell lysate or tissue extract in a 1.5 ml microcentrifuge tube, add µl of the suspended (25% v/v) IP matrix supplied with each kit. Incubate for 30 minutes at 4 C while rotating. NOTE: If the lysate was prepared from cells expressing Igs (i.e., spleen cells or cultured B cells), a preclearing step with Protein A/G agarose should also be performed 2-3 times to ensure complete removal of endogenous Igs. Pellet IP matrix via microcentrifugation at maximum speed for 30 seconds at 4 C. Without disturbing pellet, transfer desired supernatant (precleared cell lysate) to a new microcentrifuge tube. Store precleared lysate on ice and discard the pellet. Formation of the IP antibody-ip matrix complex: To a microcentrifuge tube, add µl of suspended (25% v/v) IP matrix, 1-5 µg of IP antibody and 500 µl of PBS. Optimal antibody amount should be determined by titration. Incubate at 4 C on a rotator for at least one hour. This step can be performed in parallel with the above preclearing step or performed the day before and allowed to incubate overnight at 4 C. NOTE: It is necessary that the species of the IP antibody matches the species of the IP matrix included with each ExactaCruz kit. For example, when performing an IP with a mouse antibody, it must be incubated with the Mouse IP Matrix provided (sc or sc-45042). After incubation of the IP antibody with the species specific IP matrix, pellet matrix via microcentrifugation at maximum speed for 30 seconds at 4 C. Carefully aspirate and discard supernatant. Wash pelleted matrix 2 times with 500 µl of PBS, each time repeating the above centrifugation and aspiration steps. Immunoprecipitation: After the final wash of the IP antibody-ip matrix complex, transfer lysate ( µg of total cellular protein) to the pelleted matrix and incubate at 4 C on a rotator for one hour to overnight. After incubation of the matrix and lysate, microcentrifuge at maximum speed for 30 seconds at 4 C to pellet. Aspirate and discard supernatant or alternatively keep supernatant for another IP or testing via western blot. Wash pelleted matrix 2-4 times with either RIPA Lysis Buffer: sc (more stringent) or 1x PBS (less stringent), each time repeating the above centrifugation and aspiration steps. After final wash, aspirate and discard the supernatant and resuspend pellet in µl of reducing 2x Electrophoresis Sample Buffer: sc Boil samples for 2-3 minutes. Note: The immunoprecipitated sample must be completely reduced and denatured for ExactaCruz to work properly. Perform a quick spin to pellet IP matrix and carefully load supernatant onto gel. Continue with electrophoresis as described under the Western (Immuno) Blotting procedure. 3

4 At this stage it is essential that the immunoblotting (primary) antibody matches the species specificity of the HRP conjugated ExactaCruz detection reagent which is unique for each kit. Detect the immunoblotting (primary) antibody using the corresponding HRP conjugated ExactaCruz reagent and Western Blot Luminol Reagent: sc NOTE: When using sc ExactaCruz E, the alternate immunoblotting protocol that is specific for this kit must be followed as described below in order to generate desired results. ExactaCruz E Alternate Protocol After transfer, block/wash membrane with TBST (10x TBST: sc-24953) for 1 hour, changing TBST once half way through the incubation. Dilute WB antibody with ExactaCruz E Dilution Buffer (provided), add to membrane and incubate for 1-2 hours at room temperature. After incubation, wash 3x with 1x TBST, 5 minutes per wash. Dilute ExactaCruz E Western Blot Reagent (1:1000-1:10000) with ExactaCruz E Dilution Buffer (provided), add to membrane and incubate 1-2 hours at room temperature. Wash membrane 3x with TBST, 5 minutes per wash. Wash membrane once with 1x TBS (10x TBS: sc-24951) for 5 minutes. Incubate membrane in Western Blot Luminol Reagent: sc-2048 according to Luminol data sheet. 5. IMMUNE COMPLEX PROTEIN KINASE ASSAYS NOTE: For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents. Remove medium from 100 mm cell culture plate (80 90% confluent monolayer) and wash once with PBS. Add 1 3 ml ice cold RIPA buffer (sc-24948) to cell monolayer and incubate at 4 C for 10 minutes. (NOTE: the use of RIPA buffer may not be optimal for some kinases. Composition of lysis buffer may need to be optimized to maintain active kinase.) Disrupt cells by repeated passage through a 21-gauge needle and transfer to microcentrifuge or 15 ml conical centrifuge tube. Wash cell culture plate with addition of 1.0 ml ice cold RIPA buffer, 0.5% Triton X-100 (Triton X-100: sc-29112) and combine with original extract. Pellet cellular debris at 10,000xg for 10 minutes at 4 C. Transfer supernatant to a new microcentrifuge or 15 ml conical centrifuge tube at 4 C. Transfer 1.0 ml cell extract (supernatant from above step) to a 1.5 ml microcentrifuge tube. Add 1 10 µl (i.e., µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4 C. Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Cap tubes and incubate at 4 C on a rocker platform or rotating device for 1 hour to overnight. Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4 C. Carefully aspirate and discard supernatant. Wash pellet four times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above. Suspend pellet in 20 µl of the appropriate protein kinase assay buffer (e.g., 50 mm HEPES (HEPES: sc-29097), 0.1 mm EDTA (EDTA: sc-29092), 0.01% BRIJ 35, 0.1 mg/ml BSA, 0.1% β-mercaptoethanol (β-mercaptoethanol: sc-29086), 0.15 M NaCl (NaCl: sc-29108). Buffer composition will depend upon the kinase under study. Add ng peptide substrate. Peptide substrate concentration should be determined empirically for the substrate/enzyme/cell line used. Prepare 1 ml ATP mix: 930 µl appropriate protein kinase assay buffer, 6 µl 50 mm ATP, ph 7.0, 20 µl 2.0 M MgCl2, and 44 µl [γ 32 P]-ATP [10 mci/ml]. Add 10 µl ATP mix per sample and incubate for 20 minutes at 30 C. Place on ice. Terminate the reaction by adding an equal volume of 2x electrophoresis sample buffer (sc-24945) and boil samples for 2 3 minutes. After boiling, samples may be centrifuged to pellet the agarose beads (optional); the supernatant is analyzed. Analyze samples by SDS-PAGE and autoradiography. Unused samples may be stored at -20 C. Labeled peptides can be separated from unicorporated label by acid precipitation followed by collection on a filter and radioactivity determined by scintillation counting. Researchers may also choose to analyze immobilized peptides prepared by standard methods or offered commercially. 6. IMMUNO-PEROXIDASE CELL STAINING NOTE: For a listing of cover glasses and micro slides for Immunohistochemistry. A. Tissue Culture Cells NOTE: For a listing of offered cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents, 4

5 Grow cultured cells on sterile glass cover slips or slides overnight at 37 C. Wash briefly with PBS and fix cells by one of the following procedures: 1) 5 minutes in -10 C methanol, air dry (recommended method); or 2) 2 minutes in cold acetone, air dry; or 3) 10 minutes in 1% formalin in PBS (keep wet). Wash in three changes of PBS. Optional: Incubate for 5 10 minutes in 0.1 1% hydrogen peroxide in PBS to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each. B. Frozen Tissue Sections Freeze tissue block in liquid nitrogen according to standard procedures. Block may be stored at -70 C for up to 2 weeks before sectioning. Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides. Cut 4- to 10-micron thick sections. Adhere sections to room temperature slides. Slides may be stored at -70 C. Thaw slides at room temperature prior to fixing and staining. NOTE: For a listing of mounting media for Immunohistochemistry including Clarion and Crystal Mounting Media. Fix slides in cold acetone for 10 minutes and keep refrigerated (or choose other fixation procedure). Wash in three changes of PBS. Optional: Incubate for 5 10 minutes in 0.1 1% hydrogen peroxide in PBS to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each. NOTE: For tissues containing high levels of endogenous biotin (which may result in higher background staining), we recommend following the Formalin-Fixed, Paraffin-Embedded Tissue Sections protocol, as endogenous biotin is normally destroyed in paraffin-embedded tissue. C. Formalin-Fixed, Paraffin-Embedded Tissue Sections Fix tissue sections in formalin and embed in paraffin blocks according to standard procedures. Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides. Cut 4 6 micron thick tissue sections, and apply to slides. Deparaffinize in xylenes using three changes for 5 minutes each. Hydrate sections gradually through graded alcohols: wash in 100% ethanol twice for 10 minutes each, then 95% ethanol twice for 10 minutes each. Wash in deionized H 2 O for 1 minute with stirring. Aspirate excess liquid from slides. Optional: Antigen unmasking may be performed at this point. Certain antigenic determinants are masked by formalin fixation and paraffin embedding and may be exposed by one of several methods: 1) Heat treatment (recommended method): Place slides in a container and cover with 10 mm sodium citrate buffer, ph 6.0; or with 50 mm glycine- HCl buffer (glycine: sc-29096), ph 3.5, with 0.01% (w/v) EDTA (EDTA: sc-29092). Heat at 95 C for 5 minutes. Top off with fresh buffer and heat at 95 C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H 2 O three times for 2 minutes each. Aspirate excess liquid from slides. 2) Pepsin: Incubate sections for minutes in 0.1% pepsin in 0.01 N HCl at room temperature. Wash slides several times in deionized H 2 O. Aspirate excess liquid from slides. 3) Saponin: Incubate sections for 30 minutes in 0.05% saponin in deionized H 2 O at room temperature. Wash at least three times in PBS. Aspirate excess liquid from slides. Optional: Incubate for 5 10 minutes in 0.1 1% hydrogen peroxide in deionized H 2 O to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each. D. Immunoperoxidase Staining For immunoperoxidase staining of tissue sections, we recommend the use of either the Santa Cruz Biotechnology, Inc. ABC Staining Systems or the ImmunoCruz Staining Systems. The ABC Staining Systems utilize preformed avidin-biotinylated horseradish peroxidase complex as a detection reagent, whereas the ImmunoCruz Staining Systems utilize a streptavidin-horseradish peroxidase complex. The ImmunoCruz Staining Systems include all secondary reagents in a pre-diluted, ready to use format. Complete research protocols are included with all Staining Systems; brief protocols are given below. All steps are carried out at room temperature in a humidified chamber. Allow all Staining System reagents to reach room temperature prior to use. Tissue sections should not be allowed to dry out at any time during the procedure. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagents to cover the specimens (approximately 100 µl per slide is usually adequate). ABC Staining Systems Incubate specimens for 1 hour in 1.5% normal blocking serum in PBS. Blocking serum ideally should be derived from the same species in which the secondary antibody is raised. Remove blocking serum from slides. Incubate with primary antibody for 30 minutes at room temperature or overnight at 4 C. Optimal antibody concentration should be determined by titration; recommended range is µg/ml diluted in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each. 5

6 Incubate for 30 minutes with biotin-conjugated secondary antibody as provided, or at approximately 1 µg/ml diluted in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each. Incubate for 30 minutes with avidin biotin enzyme reagent. Wash with three changes of PBS for 5 minutes each. Incubate in peroxidase substrate as provided for 30 seconds 10 minutes, or until desired stain intensity develops. Individual slides should be monitored to determine the proper development time. Wash sections in deionized H 2 O for 5 minutes. If desired, counter-stain in Gill s formulation #2 hematoxylin (sc-24973) for 5 10 seconds. Immediately wash with several changes of deionized H 2 O. Dehydrate through alcohols and xylenes as follows: Soak in 95% ethanol twice for 10 seconds each, then 100% ethanol twice for 10 seconds each, then xylenes three times for 10 seconds each. Wipe off excess xylene. Immediately add 1 2 drops of permanent mounting medium (e.g., Clarion sc-24942), cover with a glass coverslip (sc-24975) and observe by light microscopy. ImmunoCruz Staining Systems Incubate specimens for 20 minutes in 1 3 drops of serum block. Aspirate serum from slides. Dilute primary antibody in serum block to µg/ml as determined by titration. Incubate for 2 hours. Rinse with PBS then wash in PBS twice for 2 minutes each on a stir plate. Aspirate excess liquid from slides. Incubate for 30 minutes in 1 3 drops of biotinylated secondary antibody. Wash as above. Incubate for 30 minutes in 1 3 drops of HRP-streptavidin complex. Wash as above. Add 1 3 drops HRP substrate mixture. Develop for 30 seconds 10 minutes, or until desired stain intensity develops. Rinse with deionized H 2 O and transfer to a deionized H 2 O wash for 2 minutes on a stir plate. Counterstain, dehydrate and mount slides as described under ABC Staining Systems. 7. IMMUNOFLUORESCENCE CELL STAINING NOTE: For a listing of cover glasses and micro slides for Immunohistochemistry. Prepare slides as described above for immunoperoxidase staining, omitting the final step involving treatment of cells with H 2 O 2. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (approximately µl per slide is adequate). Incubate specimens with 10% normal blocking serum in PBS for 20 minutes to suppress non-specific binding of IgG. Blocking serum ideally should be derived from the same species in which the secondary antibody is raised. Wash with PBS. Incubate with primary antibody for 60 minutes. Optimal antibody concentration should be determined by titration; recommended range is µg/ml in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each. Incubate for 45 minutes with either biotin-conjugated or fluorochrome-conjugated secondary antibody diluted to 1 5 µg/ml in PBS with 1.5% 3% normal blocking serum. Optimal antibody concentration should be determined by titration. Wash with three changes of PBS. If fluorochrome-conjugated secondary antibody is used, incubate in a dark chamber and omit the next step. Incubate with streptavidin-fluorescein for 15 minutes in a dark chamber. Optimal streptavidin conjugate concentration for a given application should be determined by titration; recommended range is µg/ml in PBS. Wash extensively with PBS. Mount coverslip with aqueous mounting medium or 90% glycerol in PBS. Examine using a fluorescence microscope with appropriate filters. Store slides in a dark location at room temperature (UltraCruz Mounting Medium: sc-24941) or at 4 C (glycerol/pbs mount). 8. FLOW CYTOMETRY NOTE: For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents. Cell Surface Staining Prepare cells. 1) Lyse red blood cells. (NOTE: This will prepare enough cells for 10 samples at 100 µl/test.) Add 1 ml of whole blood to 14 ml of room temperature 1x FCM lysing solution (sc-3621). Vortex gently and incubate at room temperature for 3 5 minutes. Do NOT exceed 5 minutes. Centrifuge for 5 minutes, aspirate supernatant, and gently resuspend pellet in 5 ml of cold 1x PBS. Centrifuge again for 5 minutes, aspirate supernatant, and gently resuspend pellet in 1 ml of cold 1x PBS. 6 2) Prepare cultured cells. (NOTE: This protocol is usually performed with 50 ml of cells in media. For monolayer cells, do NOT trypsinize! Instead,

7 use a 0.2% EDTA/PBS solution. Wash cells once in 1x PBS.) Count cells using a hemocytometer. Centrifuge at 1000 rpm for 5 minutes, aspirate supernatant, and gently resuspend pellet in enough 1x PBS for a final concentration of 1 x 10 7 cells/ml. Add fluorochrome-conjugated primary antibody or isotype control to tubes. (Use 20 µl per 10 6 cells. Titrate each antibody for optimum concentration). Add 100 µl of lysed whole blood or single cell suspension to tubes. Vortex and incubate for minutes in an ice bucket. Add ~1.5 ml of FCM wash buffer (sc-3624) to each tube and mix by inversion. Centrifuge tubes at 1000 rpm for 5 minutes, aspirate supernatant, and resuspend pellet in 500 µl of 1% paraformaldehyde. Acquire and analyze data. Intracellular Staining Prepare cells by stimulating with appropriate agent, if applicable. Be sure to have an unstimulated control. Pour cell suspension into 50 ml conical tubes. Spin down cells for 5 minutes at 2000 rpm and remove media. Resuspend cells in each tube in 20 ml of room temperature 1x PBS. Perform cell count. Spin down cells for 5 minutes at 2000 rpm and remove PBS. Wash once in 50 ml of 4 C 1x PBS. Centrifuge for 5 minutes at 2000 rpm and remove PBS. Add 1 ml of 4 C FCM fixation buffer (sc-3622) for every 10 6 cells and incubate on ice for minutes. Wash cells twice in 50 ml of 4 C 1x PBS, then centrifuge and remove second wash. Add 1 ml of -20 C FCM permeabilization buffer (sc-3623) for every 10 6 cells dropwise while vortexing. Incubate on ice for 15 minutes. Spin down cells in permeabilization buffer; wash twice with 4 C FCM wash buffer (sc-3624). Centrifuge cells for 5 minutes at 2000 rpm and remove buffer. Add 1 ml of FCM wash buffer per 10 7 cells, then aliquot 100 µl of cells (10 6 ) into separate sample tubes. Stain cells intracellularly by adding 20 µl of the fluorochrome-conjugated primary antibody or isotype control to the appropriate tube and incubate for 1 hour at room temperature in the dark. NOTE: Titration of the fluorochrome-conjugated antibody should be performed for optimal results. Wash cells twice with 1 ml of FCM wash buffer then resuspend cells in 500 µl of fresh FCM wash buffer. Acquire data within 24 hours. 9. ELISA ASSAYS Coat microtiter plates with target protein diluted in 50 mm carbonate buffer at ph 9.0. Optimal concentrations should be determined by titration, but for purified antigens 50 µl per well at 1 µg/ml is usually sufficient. Incubate overnight at 4 C covered with parafilm. Remove antigen solution. Add 200 µl/well of blocking buffer (PBS containing 1% BSA and 0.02% azide) to block non-specific protein binding. Incubate for 1 2 hours at room temperature, or overnight at 4 C. Remove blocking buffer. Wash once with PBS with 0.02% azide. Damp strip wells or plates are usually stable in resealable plastic storage bags for 4 weeks at 4 C. Before using, remove excess liquid. Add test antibody samples and controls at 50 µl/well diluted in blocking buffer. Antibodies may be serially diluted for determining titer or diluted to previously determined working concentration for screening assays or antigen quantitation. Incubate 1 hour at room temperature. Wash plates three times with PBS containing 0.05% Tween-20 (Tween-20: sc-29113), removing excess liquid as above. Add 50 µl/well of alkaline phosphatase-conjugated secondary antibody diluted to 1:100 1:1000 in blocking buffer. Optimal antibody concentration is determined by titration. Incubate 1 hour at room temperature. Remove liquid in wells. Wash three times with PBS containing 0.05% Tween-20 and dry plate. Wash wells once with diethanolamine buffer (10 mm diethanolamine, 0.5 mm MgCl 2 (MgCl 2 : sc-29098), ph 9.5) and remove liquid. Dilute substrate (PNPP, sc-3720) in diethanolamine buffer to a final concentration of 1 mg/ml. Add 50 µl/well. Allow to develop for minutes or until positive control reaches an OD 405/490 of about 1.0. Stop reaction by adding 50 µl of 0.1 M EDTA (EDTA: sc-29092), ph 7.5. Read plates on microtiter plate reader at OD 405/490. 7

8 10. TRANSCRUZ GEL SUPERSHIFT ASSAYS Label oligonucleotide probe with [γ 32 P]-ATP to 50,000 cpm/ng by using polynucleotide kinase (for a listing of these reagents and more). Prepare gel shift reaction buffer as follows: 10 mm Tris (Tris: sc-3715), ph 7.5, 50 mm NaCl (NaCl: sc-29108, 1 mm dithiothreitol (DTT: sc-29089), 1 mm EDTA (EDTA: sc-29092), 5% glycerol (glycerol: sc-29095). Prepare 20 µl reaction mixture containing 3 10 µg nuclear extract and 1 µg poly di-dc in gel shift reaction buffer. Add 0.5 ng labeled oligonucleotide probe and incubate for 20 minutes at room temperature. This constitutes the control sample for detection of DNA-protein complexes. To detect an antibody supershift or block of the DNA-protein complex, prepare reaction mixture as described above, also adding 1 2 µl of the appropriate TransCruz Gel Supershift antibody per 20 µl of reaction volume. Antibody is normally added subsequent to addition of labeled oligonucleotide probe, but result may be improved by adding antibody prior to probe. Incubate at 4 C for 1 hour to overnight, or at room temperature for minutes. Resolve DNA-protein complexes by electrophoresis (25 35 ma) through a 4% polyacrylamide gel containing 50 mm Tris, ph 7.5, 0.38 M glycine (glycine: sc-29096) and 2 mm EDTA. Dry the gel and visualize by autoradiography. 11. PEPTIDE NEUTRALIZATION Blocking (neutralizing) peptides are available as negative controls for all Santa Cruz Biotechnology, Inc. affinity-purified rabbit and goat polyclonal antibodies and monoclonal antibodies raised against peptide antigens. Antibody binding to antigen may be blocked/competed by pre-absorption with the blocking peptide. Determine the highest antibody dilution at which a consistently positive result is achieved for the desired test. For example, H-Ras (259) is recommended for immunoprecipitation at 1 µg/ml but is positive at a dilution of 50 ng/ml. For blocking/competition, combine antibody (at a concentration determined by the aforementioned method) with a five-fold (by weight) excess of blocking peptide in a small volume (500 µl) of PBS. Incubate for up to 2 hours at room temperature or overnight at 4 C. Following blocking/competition, dilute antibody/peptide mixture into appropriate blocking buffer and proceed with the desired research application. 12. CHROMATIN IMMUNOPRECIPITATION (ChIP) ASSAYS NOTE: ChIP protocols vary widely. The following protocol should be suitable for most experiments. Wash cells twice with PBS at room temperature, resuspending to approximately 5x10 5 cells/ml (approximately 2x10 7 cells total). Add formaldehyde to a final concentration of 1% and incubate at room temperature for 10 minutes. Terminate cross-linking reactions by adding glycine to a final concentration of M. Pellet cells (2,000 rpm, 5 minutes) and wash once with ice cold PBS. Resuspend cells in 6 ml Lysis Buffer (sc-45000) by mixing gently. Collect crude nuclear extract by microcentrifugation at 2,000 RPM, 5 minutes. Wash again with PBS. Pellet may be frozen or processing may be continued as follows: Resuspend pellet in ~1.9 ml Lysis Buffer High Salt (sc-45001) and transfer to 2 ml microcentrifuge tube for the sonication step. Sonication conditions should be optimized since results may vary using different sonifiers. The following conditions were established by using a Sonics VC130 with a 3 mm tip probe. Sonicate on ice at power output setting = 5 6, continuous mode, 4 times at 30 second intervals. Centrifuge extract for 15 minutes, 10,000 rpm at 4 C and save supernatant (chromatin). Determine protein concentration of supernatant. For the IP step we recommend using µg protein and µl TransCruz reagent (0.2 2 µg). NOTE: Investigators may wish to consider using the primary antibody conjugated to sepharose or magnetic beads as an alternative to using secondary immunoprecipitation reagents (e.g., Protein A-Agarose) as described here. Combining primary antibodies directed to different epitopes of the same protein may be advantageous in some cases. Preclear the chromatin solution by adding 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for 30 minutes at 4º C. Centrifuge at full speed for 5 minutes at 4º C. Add primary antibody to the supernatant and incubate overnight at 4 C. Add 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for 2 hrs at 4º C. Harvest beads by centrifugations at 12,000 rpm for 20 seconds and place tube in ice. Wash beads twice with 1 ml Lysis Buffer High Salt (sc-45001). 8

9 Wash pellet four times with Wash Buffer (sc-45002). Resuspend beads in 400 µl Elution Buffer (sc-45003). Reverse cross-links by incubating tube in a 67º C water bath, mixing occasionally over two hours. Remove beads by centrifugation and continue incubating supernatant at 67º C overnight. Centrifuge for 3 minutes at 10,000 to remove any residual beads and save supernatant. To isolate DNA, extract supernatant once with 500 µl phenol/chloroform/isoamyl alcohol (25:24:1), vortex thoroughly and separate phases by centrifuging tube for 3 minutes at 14,000 rpm. Save the aqueous phase, back extract the organic phase once with 100 µl 10 mm Tris, 1 mm EDTA, ph 8.1 (TE) and pool aqueous phases. Extract pooled aqueous phase with 600 µl chloroform/isoamyl alcohol. DNA may be concentrated by using commercially available kits. 13. sirna MEDIATED INHIBITION OF GENE EXPRESSION In a six well tissue culture plate, seed 2 x 10 5 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes. Incubate the cells at 37 C in a CO 2 incubator until the cells are 60-80% confluent. This will usually take hours. NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection. Prepare the following solutions: Solution A: For each transfection, dilute 2-8 µl of sirna duplex (i.e., µg or pmols sirna) into 100 µl sirna Transfection Medium: sc Solution B: For each transfection, dilute 2-8 µl of sirna Transfection Reagent: sc into 100 µl sirna Transfection Medium: sc Peak activity should be at about 6 µl sirna Transfection Reagent. NOTE: Do not add serum and antibiotics to the sirna Transfection Medium: sc NOTE: Optimal sirna amount used for transfection may vary for each target protein and should be determined experimentally. NOTE: If a lower sirna concentration is desired, dilute sirna appropriately with sirna Dilution Buffer: sc NOTE: Although highly efficient in a variety of cell lines, sirna Transfection Reagent: sc may not be suitable for use with all cell lines. Add the sirna duplex solution (Solution A) directly to the dilute Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture minutes at room temperature. Wash the cells once with 2 ml of sirna Transfection Medium: sc Aspirate the medium and proceed immediately to the next step. For each transfection, add 0.8 ml sirna Transfection Medium to each tube containing the sirna: Transfection reagent mixture. Mix gently and overlay the mixture onto the washed cells. Incubate the cells 5-7 hours at 37 C in a CO 2 incubator. NOTE: Longer transfection times may be desirable depending on the cell line. However prolonged serum starvation may result in unwanted cell detachment or death. Add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium) without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with 1x normal growth medium. Incubate the cells for an additional hours. Aspirate the medium and replace with fresh 1x normal growth medium. Assay the cells using the appropriate protocol hours after the addition of fresh medium in the step above. NOTE: Controls should always be included in sirna experiments. Use either Control sirnas: sc-37007, sc-44230, sc-44231, sc-44232, sc-44233, sc-44234, sc-44235, sc-44236, sc or sc or Control sirna (Fluorescein Conjugates): sc-36869, sc-44239, sc or sc Each contain a scrambled sequence that will not lead to the specific degradation of any known cellular mrna. NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 µl 1x Electrophoresis Sample Buffer (sc ) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary. NOTE: For RT-PCR analysis isolate RNA using the method described by Chomczynski and Sacchi (Anal Biochem Apr;162(1): Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Chomczynski P, Sacchi N.) or a commercially available RNA isolation kit. 9

10 14. SEMI-QUANTITATIVE RT-PCR 1. cdna Synthesis: Prepare a solution containing: 1 µl oligo (dt) (500 µg/ml) 1 ng-5 µg total RNA 1 µl 10 mm dntps and add RNase-free water to a final volume of 12 µl Incubate at 70 C for 5 minutes to minimize RNA secondary structure, quick chill on ice and then add: 4 µl 5x reverse transcriptase buffer 2 µl 0.1 M DTT 1 µl RNase inhibitor Incubate at 42 C for 2 minutes to anneal primer and template. Add 1 µl reverse transcriptase (200 units) and incubate at 42 C for 50 minutes to extend the primer and then terminate the reaction by incubating at 70 C for 15 minutes. NOTE: (As an optional step add 1 µl RNase H (2 unit/µl) and incubate at 37 C for 20 minutes) 2. First PCR reaction: Prepare a solution containing: 5 µl 10x PCR buffer (with or without* MgCl 2 ) *5 µl 25 mm MgCl 2 (It may be necessary to vary the MgCl 2 concentration, 2.5 mm final concentration recommended.) 1 µl 10 mm dntp 1 µl primer pair A 1 µl Taq DNA polymerase 2 µl cdna and add water to 50 µl Incubate at 94 C for 2 minutes to denature the cdna. Perform PCR cycles. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. 3. Second PCR reaction: Prepare a solution containing: 5 µl 10x PCR buffer (with or without* MgCl 2 ) *5 µl 25 mm MgCl 2 (It may be necessary to vary the MgCl 2 concentration, 2.5 mm final concentration recommended.) 1 µl 10 mm dntp 1 µl primer pair B 1 µl Taq DNA polymerase 1 5 µl first PCR product and add water to 50 µl Incubate at 94 C for 2 minutes to denature the cdna. Perform PCR cycles. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. PCR products are separated on agarose gels and visualized by ethidium bromide staining. 15. GENERAL SOLUTIONS NOTE: For a listing of reagents required to prepare the following solutions. Blotto A (for general use): 1x TBS, 5% milk, 0.05% Tween-20. Available pre-made (sc-2333). Blotto B (for use with anti-phosphotyrosine antibodies): 1x TBS, 1% milk, 1% BSA, 0.05% Tween-20. In some cases, milk may be left out entirely, but this will result in somewhat higher backgrounds. Available pre-made (sc-2335). For all phospho-specific antibodies, add 0.01% (v/v) of each Phosphatase Inhibitor Cocktail A and B (sc and sc-45045) to inhibit phosphatase activity. Diaminobenzidine tetrahydro-chloride (DAB): Dissolve 5 mg DAB in 100 ml 100 mm Tris-HCl, ph 7.6, and add 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB solution daily. Electrophoresis sample buffer (2x): Mix 1.0 ml glycerol, 0.5 ml β-mercaptoethanol, 3.0 ml 10% SDS, 1.25 ml 1.0 M Tris-HCl, ph 6.7 and 1 2 mg bromophenol blue. Store frozen in small aliquots. Alternatively, make buffer without β-mercaptoethanol and store at room temperature. Add β-mercaptoethanol just before using. Available pre-made (sc-24945). Phosphate buffered saline (1x PBS): 9.1 mm dibasic sodium phosphate, 1.7 mm monobasic sodium phosphate and 150 mm NaCl. Adjust ph to 7.4 with NaOH. Available pre-made in liquid (sc-24946) and powder (sc-24947) forms. 10

11 RIPA buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add protease inhibitors at time of use from the following stock solutions. Available pre-made (sc-24948). 1) 10 mg/ml PMSF (sc-3597) in isopropanol (add at 10 µl/ml RIPA) 2) Aprotinin (sc-3595) (add at 50 KIU/ml RIPA) 3) 100 mm sodium orthovanadate (sc-3540) in frozen aliquots (add at 10 µl/ml RIPA) Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled H 2 O. Tris buffered saline (1x TBS): 10 mm Tris-HCl, ph 8.0; 150 mm NaCl (Tris: cat # sc-3715). Available pre-made in liquid (sc-24951) form. 11