TWC BioSearch International

INTRODUCTION

1. immunoprecipitation,
2. Western (immuno-) blotting,
3. agarose conjugate/Western blots,
4. immune complex protein kinase assays,
5. immunoperoxidase cell staining,
6. immunofluorescence,
7. ELISA assays,
8. TransCruz^TM gel supershift assays,
9. methods for use of peptides to neutralize antibody activity and
10. preparation of solutions common to many of the procedures.

1. IMMUNOPRECIPITATION

1. Incubate cultured cells (80­90% confluent monolayer in 100 mm petri dish, or approximately 2 to 5 x 107 suspension cells in flask) in methionine-free medium containing 5% dialyzed fetal calf serum for 1 hour at 37° C. (Same procedure can be used for cells labeled with other radioactive amino acids (e.g. ¹⁴C or ³H), or with ³²P-orthophosphate. Cell labeling must be carried out in medium lacking the relevant amino acid, or in phosphate-free medium.)

2. Remove medium, replace with 3 ml methionine-free medium containing 5% dialyzed fetal calf serum and 100 µCi/ml ³⁵S-methionine. Incubate 1 hour at 37° C. For some proteins a longer labeling period (up to 18 hours) is preferable.

3. Carefully remove radioactive medium with Pasteur pipette and wash cell monolayer with PBS.

4. Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4° C for 10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a 15 ml conical centrifuge tube.

5. Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube.

6. Wash petri dish with additional 1.0 ml ice cold RIPA buffer and combine with original extract.

7. Pellet cellular debris by centrifugation at 3,000 rpm at 4° C for 15 minutes. Transfer supernatant to 15 ml conical centrifuge tube and preclear lysate by adding 1.0 µg normal mouse, rat or rabbit IgG, as appropriate, together with 20 µl of agarose conjugate. Pellet by centrifugation at 1,500 rpm at 4° C for 5 minutes and transfer supernatant (cell lysate) to 15 ml conical centrifuge tube at 4° C.

8. Transfer 1.0 ml extract to a 1.5 ml microcentrifuge tube; add 1­10 µl (i.e., 0.1­1 µg) monoclonal or polyclonal antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4° C. Alternatively, if antibody conjugate is available, add 5 µl of packed beads and skip the next step.

9. Add 20 µl agarose conjugate (Protein A-Agarose, Protein G PLUS-Agarose or Protein A/G PLUS-Agarose, see Table below). Cap tubes and incubate at 4° C on rocker platform or rotating device for 1 hour to overnight.

10. Collect immunoprecipitates by centrifugation at 2500 rpm for 5 minutes at 4° C.

11. Carefully aspirate and discard radioactive supernatant; wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), repeating centrifugation step as above.

12. After final wash, aspirate and discard supernatant, resuspend pellet in 40 µl electrophoresis sample buffer.

13. Boil samples for 3 minutes and analyze (20 µl aliquots) by SDS-PAGE, or store at -20° C. After boiling, samples may be centrifuged to pellet the agarose beads (optional), followed by SDS-PAGE analysis of the supernatant.

Immunoprecipitation Reagents

product	specificity	cat #	volume	# reactions
Protein A-Agarose	mouse IgG2a and IgG2b rabbit polyclonal Abs	sc-2001	2.0 ml	100
Protein G PLUS-Agarose	mouse IgG1 & IgG3 rabbit and goat polyclonal Abs rat IgG1, IgG2a, IgG2b and IgG2c	sc-2002	2.0 ml	100
Protein A/G PLUS-Agarose	all of the above Abs	sc-2003	2.0 ml	100
Glutathione-Agarose	GST fusion proteins	sc-2009	1.0 ml	100

Immunoprecipitation agarose conjugates are pre-blocked with BSA to reduce non-specific immunoglobulin binding and are provided at a concentration (0.5 ml agarose/2.0 ml) suitable for use at 20 µl per immunoprecipitation reaction.

2. WESTERN (IMMUNO-) BLOTTING

A. Sample Preparation

Monolayer cells

1. Remove medium and rinse 100 mm petri dish with PBS at room temperature. All following steps should be done on ice or at 4° C using ice cold buffers.

2. Add 0.6 ml of RIPA buffer (with freshly added inhibitors) to a 100 mm petri dish. Scrape cells with a cell scraper. Using a syringe fitted with a 21 gauge needle, transfer the lysate to a microcentrifuge tube.

3. Wash the plate once with 0.3 ml of RIPA buffer, combine with first lysate, and pass through the 21 gauge needle to shear the DNA. Add 10 µl of 10 mg/ml PMSF stock. Incubate 30­60 minutes on ice.

4. Microcentrifuge cell lysate at 15,000xg for 20 minutes at 4° C. The supernatant liquid is the total cell lysate.

   Suspension cells

5. Collect approximately 2.0 X 10⁷ cells by low speed centrifugation at room temperature for five minutes. Carefully remove medium.

6. Wash the pellet with PBS at room temperature, and again collect by low speed centrifugation. Carefully remove PBS.

7. Add 1.0 ml of ice cold RIPA buffer with freshly added inhibitors. Mix gently with a pipette and incubate on ice for 30 minutes.

8. Further disrupt and homogenize cells by passage through 21 gauge needles, dounce homogenization or sonication, taking care not to raise the temperature of the lysate. Add 10 µl of 10 mg/ml PMSF stock. Incubate 30 minutes on ice.

9. Transfer to microcentrifuge tubes, centrifuge at 15,000xg for 20 minutes at 4° C. The supernatant fluid is the total cell lysate.

Tissue samples

1. Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue slices very thin and can be thawed in lysis buffer containing inhibitors. Use 3 ml of ice cold RIPA buffer per gram of tissue.

2. Further disrupt and homogenize tissue with a dounce homogenizer or a polytron device, maintaining temperature at 4° C throughout all procedures. Add 30 µl of 10 mg/ml PMSF stock per gram of tissue and incubate on ice for 30 minutes.

3. Transfer to microcentrifuge tubes, centrifuge at 15,000xg for 20 minutes at 4° C. Remove supernatant and centrifuge again. The supernatant fluid is the total cell lysate. (Sometimes a longer centrifugation is necessary to obtain a clarified lysate.)

B. Electrophoresis

1. Mix sample (40­60 µg whole cell lysate, 10­20 µg nuclear extract, or 10­20 ng purified protein) with an equal volume of electrophoresis sample buffer and boil for 90 seconds.

2. Load up to 10 µl of lysate per 1.0 mm of well width for gels of 0.75 mm thickness.

3. In one lane of the gel, you may use Cruz Marker^TM molecular weight standards (cat # 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^TM compatible secondary antibodies (see Table on page 2), internal standard bands will appear when the probed blot is exposed to chemiluminescence or color developer.

4. Carry out electrophoresis according to standard protocols.

5. Transfer proteins from the gel to nitrocellulose membrane using an electroblotting apparatus according to the manufacturer's protocols.

6. Block non-specific binding by soaking membrane in Blotto (either Blotto A or Blotto B, see page 7) for a minimum of 15 minutes at room temperature. Alternatively, the membrane may be blocked at 4° C overnight in a covered container, using Blotto without Tween.

7. Incubate in primary antibody at 0.1 µg/ml to 1.0 µg/ml in Blotto for 45 minutes and wash three times for 5 minutes each with TBS, .05% Tween-20.

8. Incubate for 30 minutes with horseradish peroxidase- (HRP-) conjugated secondary antibody, or alkaline phosphatase (AP)-conjugated secondary antibody (see Table on page 2), diluted to 1:500­1:5000 in Blotto. If high backgrounds are observed, reduce secondary antibody concentration. If Cruz Marker^TM molecular weight standards are used in the gel, the Cruz Marker^TM compatible secondary antibodies must be used (see Table on page 2).

9. Wash three times for 5 minutes each with TBS, .05% Tween-20, and one time for 5 minutes with TBS.

10. Incubate membrane in Luminol ECL Reagent (cat # sc-2038) according to Luminol data sheet or visualize proteins using standard protocols. If Luminol is used for visualization, HRP-conjugated secondary antibody must be used.

Western Blotting Secondary Antibodies: Conventional and Cruz Marker^TMCompatible

product	conventional catalog #	Cruz MarkerTM catalog #	conjugate	amount
anti-rabbit IgG-HRP	sc-2004	sc-2030	peroxidase	200 µg
anti-mouse IgG-HRP	sc-2005	sc-2031	peroxidase	200 µg
anti-rat IgG-HRP	sc-2006	sc-2032	peroxidase	200 µg
anti-goat IgG-HRP	sc-2020	sc-2033	peroxidase	200 µg
anti-rabbit IgG-AP	sc-2007	sc-2034	alkaline phosphatase	200 µg
anti-mouse IgG-AP	sc-2008	sc-2047	alkaline phosphatase	200 µg
anti-rat IgG-AP	sc-2021	sc-2036	alkaline phosphatase	200 µg
anti-goat IgG-AP	sc-2022	sc-2037	alkaline phosphatase	200 µg

3. AGAROSE CONJUGATE/WESTERN BLOTS

1. Prepare a total cell lysate as described under Western blot procedure (see above).

2. To 1 ml of total cell lysate, add 5 µl of antibody agarose conjugate and incubate at 4° C for 1 hour to overnight with mixing (such as end over end rotation).

3. Alternatively, where antibody agarose conjugate is not available, incubate 1 ml total cell lysate with 10 µl (i.e., 1.0 µg) of primary antibody for 1 hour at 4° C, then add 20 µl of Protein A-Agarose, Protein G PLUS-Agarose, or Protein A/G PLUS-Agarose (see Table on page 1) and incubate with mixing for 1 hour to overnight at 4° C.

4. Wash pellet 4 times with either RIPA buffer or PBS with NaCl adjusted to 1.0 M, each time collecting agarose by centrifuging at 2500 rpm for 5 min at 4° C. Aspirate wash carefully and discard.

5. After final wash, add 40 µl of electrophoresis sample buffer to agarose pellet and boil for 90 seconds.

6. Load up to 5 µl of sample per 1.0 mm well width for gels of 0.75 mm thickness. Note: If Protein A-Agarose or Protein G PLUS-Agarose is used, there will be a very large band at 55K which is the heavy chain of the primary antibody. There may be a heavy chain band even with primary antibody agarose conjugates, but it will be much less prominent.

7. Continue as described under Western Blotting.

4. IMMUNE COMPLEX PROTEIN KINASE ASSAY

1. Remove medium from cells (80­90% confluent monolayer) and wash once with PBS.

2. Add 3 ml ice cold RIPA buffer to cell monolayer (100 mm petri dish) and incubate at 4° C for 10 minutes.

3. Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to 15 ml conical centrifuge tube.

4. Wash petri dish with addition of 1.0 ml ice cold RIPA buffer, 0.5% Triton X-100 and combine with original extract.

5. Pellet cellular debris at 3,000 rpm at 4° C for 15 minutes and transfer supernatant to 15 ml conical centrifuge tube at 4° C.

6. Transfer 1.0 ml disrupted cell extract to a 1.5 ml microcentrifuge tube; add 1­10 µl (i.e., 0.1­1 µg) monoclonal or polyclonal antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4° C.

7. Add 20 µl agarose conjugate (Protein A-Agarose, Protein G PLUS-Agarose or Protein A/G PLUS-Agarose; see Table on page 1).

8. Cap tubes and incubate at 4° C on rocker platform or rotating device for 1 to 24 hours.

9. Collect immunoprecipitates by centrifugation at 2500 rpm for 5 minutes at 4° C.

10. Carefully aspirate and discard supernatant, wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), repeating centrifugation step as above.

11. Suspend pellet in 20 µl of the appropriate protein kinase assay buffer (e.g., 50 mM HEPES, 0.1 mM EDTA, 0.01% Brij 35, 0.1 mg/ml BSA, 0.1% -mercaptoethanol, 0.15 M NaCl).

12. Add peptide substrate at 10­1000 ng. Peptide concentration should be determined empirically for the substrate/enzyme/cell line used.

13. Add 10 µl ATP mix (930 µl appropriate buffer for particular kinase assay, 6 µl 50 mM ATP, pH 7.0, 20 µl 2.0 M MgCl2, and 44 µl [³²P]-ATP [10 mCi/ml]). Incubate 20 minutes at 30° C. Place on ice.

14. Terminate the reaction by adding SDS-PAGE sample buffer and boil samples for 3 minutes. After boiling, samples may be centrifuged to pellet the agarose beads (optional), discard pellet and save supernatant. Analyze samples by polyacrylamide gel electrophoresis and autoradiography. Terminated samples may be stored at -20° C. If a small peptide substrate is used in the kinase assay, the sample must be analyzed by scintillation counting or other standard methods, rather than by SDS-PAGE.

5. IMMUNOPEROXIDASE CELL STAINING

A. Tissue Culture Cells

1. Grow cultured cells on sterile glass cover slips or slides overnight at 37° C. Rinse briefly with PBS and fix cells by one of the following procedures:
   1. 10 minutes in 1% formalin in PBS (keep wet); or
   2. 5 minutes in -10° C methanol, air dry; or
   3. 2 minutes in cold acetone, air dry.

2. Rinse in three changes of PBS and incubate for 5 to 10 minutes in 0.1­1% hydrogen peroxide in PBS to quench endogenous peroxidase activity (optional). Rinse in PBS twice for 5 minutes each.

B. Frozen Tissue Sections

1. Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Alternatively, pre-treated slides are available.

2. Cut 4 to 8 micron thick cryostat sections of tissue block (frozen in isopentane precooled in liquid nitrogen, embedded in OCT compound in cryomoids, and stored at -70° C).

3. Allow frozen sections to come to room temperature (30 minutes).

4. Fix slides in cold acetone for 10 minutes, keep refrigerated (or choose other fixation procedure). Rinse in three changes of PBS.

5. Incubate for 5 to 10 minutes in 0.1­1% hydrogen peroxide in PBS to quench endogenous peroxidase activity (optional). Rinse in PBS twice for 5 minutes each.

C. Formalin-Fixed, Paraffin-Embedded Tissue Sections

1. Clean glass slides with 95% ethanol, treat with subbing solution, and air dry. Alternatively, pre-treated slides are available.

2. Cut tissue sections using microtome, and apply to slides. Deparaffinize in xylene, using three changes, 5 minutes each. Hydrate sections gradually through graded alcohols, for example, soak in 100% ethanol twice for 10 minutes each, then 95% ethanol twice for 10 minutes each. Rinse once in distilled H2O for 5 minutes.

3. Incubate for 15 minutes in 0.1­1% hydrogen peroxide to quench endogenous peroxidase activity (optional). Rinse in PBS twice for 5 minutes each.

   NOTE: Antigen unmasking may be performed at this point. Certain antigenic determinants are masked by formalin fixation and paraffin embedding. In such cases the antigens often may be exposed by heat treatment, pepsin digestion or treatment with saponin. Pretreatment with saponin is recommended in cases in which enzymatic digestion is not required or can be used subsequent to pepsin digestion.

   1. Heat treatment: Place slides in a container and cover with 10 mM citrate buffer, pH 6.0; or with 50 mM glycine-HCl buffer, pH 3.5, containing 0.01% (w/v) EDTA. Microwave for 5 minutes. Change to fresh buffer, microwave for 5 minutes. Remove buffer and allow slides to cool. Rinse in PBS.

   2. Pepsin: Incubate sections for 30 minutes in 2­10 µg/ml pepsin in 0.01 N HCl buffer, pH 2.5, at room temperature; wash slides several times in distilled H2O.

   3. Saponin: Incubate sections for 30 minutes in 2­10 µg/ml saponin in distilled H2O, at room temperature; wash at least 3 times in PBS.

D. Immunoperoxidase Staining

1. Incubate specimens for 1 hour in 1.5% normal blocking serum in PBS, as provided in Santa Cruz Biotechnology Immunoperoxidase Staining Kits (see Table below), to suppress non-specific binding of IgG. Carry out this and subsequent incubations at room temperature or at 4° C in humidified chamber. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (approximately 100 µl per slide is usually adequate). Remove blocking serum from slides.

2. Incubate with primary antibody for 30 minutes at room temperature, or overnight at 4° C. Optimal antibody concentration for a given application should be determined by titration; usual range is 0.1 to 2.0 µg/ml diluted in normal blocking serum in PBS. Wash with three changes of PBS, 5 minutes each.

3. Incubate for 30 minutes with biotin-conjugated secondary antibody, as provided in Santa Cruz Biotechnology Immunoperoxidase Staining Kit. Wash with three changes of PBS, 5 minutes each.

4. Incubate for 30 minutes with avidin biotin enzyme reagent as supplied in Santa Cruz Biotechnology Immunoperoxidase Staining Kit. Wash with three changes of PBS, 5 minutes each.

5. Rinse in 1% Triton X-100/PBS for 30 seconds, then rinse once in water and blot slides dry.

6. Incubate in DAB solution for 10 seconds to 5 minutes until brown staining is visible. Individual slides should be monitored to determine the proper development time. Rinse in H2O.

7. If desired, counterstain in hematoxylin 10 minutes. Remove excess counterstain by dunking slides once in 0.25% HCl and 50% methanol, rinse in distilled H2O, and incubate for 1 minute in tap water. Rinse slides in distilled H2O three times.

8. Dehydrate through alcohols and xylene as follows: Soak in 95% ethanol twice for 10 seconds each, then 100% ethanol twice for 10 seconds each, then xylene twice for 10 seconds each. Let slides dry. Mount coverslip using a permanent mounting medium (e.g., Permount) and observe by light microscopy.

   NOTE: One note of caution in use of the Santa Cruz Biotechnology Immunoperoxidase Staining Kit is that frozen sections may sometimes have relatively high levels of endogenous biotin that can result in high background staining. The endogenous biotin is normally destroyed in paraffin-embedded tissue. A second problem that may be encountered pertains to the use of mouse or rat monoclonal antibodies to stain murine tissues with relatively high levels of endogenous immunoglobulin such as in spleen or lymph node tissues. This can cause non-specific binding of goat anti-mouse or anti-rat secondary antibodies such as those used in the Santa Cruz Biotechnology Immunoperoxidase Staining Kit.

6. IMMUNOFLUORESCENCE CELL STAINING

1. Prepare slides as described above for immunoperoxidase staining omitting the final step involving treatment of cells with hydrogen peroxide to quench endogenous peroxidase activity.

2. Incubate specimens with 10% normal serum in PBS for 20 minutes to suppress non-specific binding of IgG (optional step). Carry out this and all subsequent incubations at room temperature in a humidified chamber. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (approximately 100 to 500 µl per slide is usually adequate). Wash with PBS.

3. Incubate with primary antibody for 60 minutes. Optimal antibody concentration should be determined by titration (0.1 to 2.0 µg/ml in PBS-BSA is usually adequate). Wash with three changes of PBS, 5 minutes each.

4. Incubate with biotin-conjugated secondary antibody (see Table on page 5) for 45 minutes. Optimal antibody concentration (2 to 5 µg/ml in PBS) should be determined by titration. Wash with three changes of PBS. Alternatively incubate in fluorescein-conjugated secondary antibody (see Table on page 5) (10­20 µg/ml) and omit the next step. Wash with three changes of PBS.

5. Incubate with Streptavidin-fluorescein (cat # sc-2016) for 15 minutes in a dark chamber. Optimal Streptavidin conjugate concentration for a given application should be determined by titration. Usual range: 10 to 20 µg/ml in PBS. Wash extensively with PBS.

6. Mount coverslip with aqueous mounting medium or 90% glycerol/PBS.

7. Examine using a fluorescence microscope with appropriate filters. Store slides in the dark at room temperature (semi-permanent mounts) or at 4° C (glycerol/PBS).

7. ELISA PROCEDURE

1. Coat microtiter plates with target protein diluted in 50 mM carbonate buffer at pH 9.0. Optimum concentrations should be determined by titration, but for purified antigens on Immulon II plates (Dynatech Laboratories, Inc.) 50 µl per well at 1 µg/ml is usually sufficient. Incubate overnight at 4° C covered with parafilm.

2. Remove antigen solution by flicking and slap plate dry. Add 200 µl/well of Blocking buffer (PBS containing 1% BSA and 0.02% azide) to block non-specific protein binding. Incubate 1-2 hours at room temperature, or overnight at 4° C.

3. Remove Blocking buffer by flicking and slap plate dry. Wash once with PBS with 0.02% azide. Damp strip wells or plates are usually stable in ziplock bags for 4 weeks at 4° C. Before using, slap plate dry again.

4. 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.

5. Remove samples by flicking and wash three times with PBS containing .05% Tween-20. Slap plate to remove moisture.

6. Add 50 µl/well of alkaline phosphatase conjugated secondary antibody (see Table on page 2) diluted 1:250 in Blocking buffer. Incubate 1 hour at room temperature.

7. Remove reagent by flicking. Wash three times with PBS containing .05% Tween-20 and slap dry ­ make sure bottoms of plates are dry.

8. Wash wells once with diethanolamine buffer (10 mM diethanolamine, 0.5 mM MgCl2, pH 9.5), slap plate dry.

9. Dilute substrate in diethanolamine buffer to a final concentration of 1 mg/ml (i.e., 1 tablet of Sigma 104 phosphatase substrate per 5 ml buffer). Add 50 µl/well. Allow to develop for 10-20 minutes or until positive control reaches an OD405/490 of about 1.0. Stop reaction by adding 50 µl of 0.1 M EDTA (pH 7.5). Read plates on microtiter plate reader at 405/490.

8. TRANSCRUZ^TM GEL SUPERSHIFT ASSAYS