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Antagene Inc. Research Protocols

Antagene Inc. recommends the following procedures and secondary reagents for use with our primary antibodies:

(1) Western blotting
(2) Immunoprecipitation
(3) Immunoprecipitation/Western blots
(4) Immune complex protein kinase assays
(5) Immunoperoxidase cell staining
(6) Immunofluorescence cell staining
(7) ELISA assays
(8) Methods for the use of peptides to neutralize antibody activity
(9) Preparation of solutions

1. Western Blotting

A.Sample Preparation
Monolayer cells
Remove medium and rinse 100 mm cell culture plate with PBS at room temperature. All the following steps should be done on ice or at 4C using ice cold buffers.
Add 0.6 ml of RIPA buffer (with freshly added inhibitors) to a 100 mm cell culture plate. Scrape plate with a cell scraper. Using a syringe fitted with a 21 gauge needle, transfer the lysate to a microcentrifuge tube.
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 of 10 mg/ml PMSF stock. Incubate 30-60 minutes on ice.
Microcentrifuge cell lysate at 10,000xg for 10 minutes at 4 C. The supernatant fluid is the total cell lysate.

Suspension cells
Collect approximately 2.0 x 10⁷ cells by low speed centrifugation at room temperature for five minutes. Carefully remove medium.
Wash the pellet with PBS at room temperature, and again collect by low speed centrifugation. Carefully remove PBS.
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.
Further disrupt and homogenize cells by passage through 21 gauge needle, dounce homogenization or sonication, taking care not to raise the temperature of the lysate. Add 10 �l of 10 mg/ml PMSF stock. Incubate 30 minutes on ice.
Transfer to microcentrifuge tubes and centrifuge at 10,000xg for 10 minutes at 4� C. The supernatant fluid is the total cell lysate.

Tissue samples
Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue can be sliced very thinly and thawed in lysis buffer containing inhibitors. Use 3 ml of ice cold RIPA buffer per gram of tissue.
Further disrupt and homogenize tissue with a dounce homogenizer or a polytron device, maintaining temperature at 4� C throughout all procedures. Add 30 �l of 10 mg/ml PMSF stock per gram of tissue and incubate on ice for 30 minutes.
Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 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
Mix sample (40-60 �g whole cell lysate, 10-20 �g nuclear extract, or 10-20 ng purified protein per lane) with an equal volume of 2x electrophoresis sample buffer and boil for 2-3 minutes.
Unused samples may be stored at -20� C for up to several months.
Load up to 10 �l of lysate per 1.0 mm of well width for gels of 0.75 mm thickness.
Load 2 �l/well for 0.75 mm gels and 5 �l/well for 1.5 mm gels. When used secondary antibodies (see Western Blotting Secondary Antibodies table), internal standard bands will appear when the probed blot is exposed to chemiluminescence or color developer.
Carry out electrophoresis according to standard protocols.
Transfer proteins from the gel to nitrocellulose membrane using an electroblotting apparatus according to the manufacturer's protocols.

C. Immunoblotting
Block non-specific binding by incubating membrane in 5% fat-free milk for 30-60 minutes at room temperature. Alternatively, the membrane may be blocked at 4� C overnight in a covered container, using 5% fat-free milk without Tween-20.
Incubate in primary antibody diluted in PBS pH7.2 for 1 hour at room temperature. Optimal antibody concentration should be determined by titration. We recommend a starting dilution of 0.5-2.0 �g/ml PBS pH7.2; final optimal concentration may be as low as 0.1 �g/ml. Wash membrane three times for 5 minutes each with TBS, 0.05% Tween-20.
Incubate for 45 minutes at room temperature with horseradish peroxidase (HRP) conjugated secondary antibody, or alkaline phosphatase (AP) conjugated secondary antibody (see Western
Blotting Secondary Antibodies table), diluted to 1:500-1:2000 in PBS pH7.2. If high backgrounds are observed, secondary antibody should be diluted further, as low as 1:20,000 in some cases.
Wash membrane three times for 5 minutes each with TBS, 0.05% Tween-20, and once for 5 minutes with TBS.
Incubate membrane in Chemiluminescence Luminol Reagent according to Luminol data sheet, or visualize proteins using standard protocols. If Luminol is used for visualization, HRP-conjugated secondary antibody must be used.

2. Immunoprecipitation

Incubate cultured cells (80-90% confluent monolayer in 100 mm cell culture plate, or approximately 2-5 x 10⁷ suspension cells in flask) in methionine-free medium containing 5% dialyzed fetal calf serum for 1 hour at 37� C. The 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.
Remove medium and replace with 3 ml methionine-free medium containing 5% dialyzed fetal calf serum and 100 �Ci/ml ³⁵S-methionine. Incubate 1 hour at 37� C. For some proteins a longer labeling period (up to 18 hours) is preferable.
Carefully remove radioactive medium with Pasteur pipette and wash cell monolayer with PBS.
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.
Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube.
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 15 ml conical centrifuge tube on ice. Preclear lysate by adding 1.0 �g of the appropriate control IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host species of the primary antibody), together with 20 �l of resuspended volume of the appropriate 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 2,500 rpm (approximately 1,000xg) for 5 minutes at 4� C.
Transfer supernatant (cell lysate) to a fresh 15 ml conical centrifuge tube on ice.
Transfer 1 ml of the above cell lysate, or approximately 100-500 �g total cellular protein, to a 1.5 ml microcentrifuge tube. Add 1-10 �l (i.e., 0.2-2 �g) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4� C. Alternatively, if antibody agarose conjugate is available, add 5 �l (i.e., 10 �g) packed beads and incubate at 4� C for 1 hour to overnight with mixing; skip the next step.
Add 20 �l of resuspended volume of the appropriate agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose, see table below). 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 radioactive supernatant.
Wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.
After final wash, aspirate and discard supernatant and resuspend pellet in 40 �l of 1x electrophoresis sample buffer.
Boil samples for 2-3 minutes and analyze 20 �l aliquots by SDS-PAGE 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.

3. Immunoprecipitation/Western Blots

Prepare a total cell lysate as described under Western blot procedure (section 1).
Preclear whole cell lysate (optional step) as follows. To approximately 1 ml of whole cell lysate, add 0.25 �g of the appropriate control IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host species of the primary antibody), together with 20 �l of resuspended volume of the appropriate agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose, see table above). Incubate at 4� C for 30 minutes.
Pellet beads by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4� C. Transfer supernatant (cell lysate) to a fresh 1.5 ml microcentrifuge tube at 4� C.
To 1 ml of the above cell lysate, or approximately 100-500 �g of total cellular protein, add 10 �g of primary antibody agarose conjugate (i.e. 5 �l volume of packed beads) and incubate at 4� C for 1 hour to overnight with mixing such as end over end rotation.
Alternatively, if primary antibody agarose conjugate is not available, incubate 1 ml cell lysate with 1-10 �l (i.e., 0.2-2 �g) primary antibody (optimal antibody concentration should be determined by titration) for 1 hour at 4� C. Add 20 �l of resuspended volume of the appropriate agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose, see table above). Cap tubes and incubate at 4� C on a rocker platform or rotating device for 1 hour to overnight.
Collect pellet by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4� C. Carefully aspirate and discard supernatant.
Wash pellet 4 times with either RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.
After final wash, aspirate and discard supernatant and resuspend pellet in 40 �l of 1x electrophoresis sample buffer.
Boil samples for 2-3 minutes. Load up to 5-10 �l 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 (section 1).

NOTE: Depending on the secondary antibody that is used, bands may appear on the blot at 55 and 27 kDa which are the heavy and light IgG chains of the primary antibody. These immunoglobulin bands will be less pronounced if a primary antibody agarose conjugate is used in the above procedure.

4. Immune Complex Pretien Kinase Assays

� Remove medium from 100 mm cell culture plate (80-90% confluent monolayer) and wash once with PBS.
� Add 3 ml ice cold RIPA buffer 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 aspiration through a 21 gauge needle and transfer to 15 ml conical centrifuge tube.
� Wash cell culture plate with addition of 1.0 ml ice cold RIPA buffer, 0.5% Triton X-100 and combine with original extract.
� Pellet cellular debris at 10,000xg for 10 minutes at 4� C. Transfer supernatant to a fresh 15 ml conical centrifuge tube at 4� C.
� Transfer 1.0 ml disrupted cell extract (supernatant from above step) to a 1.5 ml microcentrifuge tube. Add 1-10 �l (i.e., 0.2-2 �g) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4� C.
� Add 20 �l of resuspended volume of the appropriate agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose, see Immunoprecipitation Reagents table). 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 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.
� Suspend pellet in 20 �l 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% b-mercaptoethanol, 0.15 M NaCl). Buffer composition will depend upon the kinase under study.
� Add peptide substrate at 10-1000 ng. Peptide substrate concentration should be determined empirically for the substrate/enzyme/cell line used.
� Prepare 1 ml ATP mix: 930 �l appropriate protein kinase assay buffer, 6 �l 50 mM ATP, pH 7.0, 20 �l 2.0 M MgCl2, and 44 �l [³²P]-ATP [10 mCi/ml]. Add 10 �l 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 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. 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
� 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 in OCT block in liquid nitrogen according to standard procedures. The frozen 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 cryostat sections of tissue block. Adhere sections to room temperature slides. Slides may be stored at -70� C. Thaw slides at room temperature prior to fixing and staining.
� Fix slides in cold acetone for10 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: Frozen sections may have relatively high levels of endogenous biotin that can result in high background staining. The 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 using microtome, 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 H₂O for 1 minute on stir plate. 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, pH 3.5, with 0.01% (w/v) EDTA. 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 H₂O three times for 2 minutes each. Aspirate excess liquid from slides.
2.Pepsin: Incubate sections for 10-20 minutes in 0.1% pepsin in 0.01 N HCl buffer, pH 2.5, at room temperature. Wash slides several times in deionized H₂O. Aspirate excess liquid from slides.
3.Saponin: Incubate sections for 30 minutes in 0.05% saponin in deionized H₂O 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 H₂O to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each.

NOTE: Antagene Inc's antibodies are only suggested for use on formalin-fixed, paraffin-embedded tissue sections if the catalog description states "immunohistochemistry (including paraffin-embedded sections)".

D. Immunoperoxidase Staining
� For immunoperoxidase staining of tissue sections, we recommend the use of the Vector Labs ABC Staining Systems. The ABC Staining Systems utilize preformed avidin-biotinylated horseradish peroxidase complex as a detection reagent, whereas the Vector Lab�s Staining Systems utilize a streptavidin-horseradish peroxidase complex. The Vector Lab�s Staining Systems include all secondary reagents in a pre-diluted, ready to use format, and are also available with pre-diluted primary antibody included. 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 �l 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 0.5-5.0 �g/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 biotin-conjugated secondary antibody as provided, or at approximately 1 �g/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 H₂O for 5 minutes. If desired, counterstain in Gill's formulation #2 hematoxylin for 5-10 seconds. Immediately wash with several changes of deionized H₂O.
� 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., Permount), cover with a glass coverslip and observe by light microscopy.

Vector Lab�s Staining Systems
� Incubate specimens for 20 minutes in 1-3 drops of serum block. Aspirate serum from slides.
� Immediately add 1-3 drops of pre-diluted primary antibody. If using a Staining System that does not include a pre-diluted primary antibody, dilute primary antibody in serum block to 0.5-5.0 �g/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 H₂O and transfer to a deionized H₂O wash for 2 minutes on a stir plate.
� Counterstain, dehydrate and mount slides as described under ABC Staining Systems.

6. Immunofluorescence Cell Staining

� Prepare slides as described above for immunoperoxidase staining, omitting the final step involving treatment of cells with hydrogen peroxide.
� Use suction to remove reagents after each step, but avoid drying of specimens between steps. � Use sufficient reagent to cover the specimen (approximately 100-500 �l per slide is usually 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 0.1-2.0 �g/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 fluorescein-conjugated secondary antibody (see Secondary Antibodies for Immunohistochemical staining table) diluted to 1-5 �g/ml in PBS with 1.5% normal blocking serum. Optimal antibody concentration should be determined by titration. Wash with three changes of PBS. If fluorescein-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 10-20 �g/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 (semi-permanent mount) or at 4� C (glycerol/PBS mount).

7. 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 on Immulon II plates (Dynatech Laboratories, Inc.) 50 �l per well at 1 �g/ml is usually sufficient. Incubate overnight at 4� C covered with parafilm.
� Remove antigen solution by flicking and slap plate dry. Add 200 �l/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 by flicking and slap plate dry. 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, slap plate dry again.
� Add test antibody samples and controls at 50 �l/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.
� Remove samples by flicking and wash three times with PBS containing 0.05% Tween-20. Slap plate to remove moisture.
� Add 50 �l/well of alkaline phosphatase conjugated secondary antibody (see Western Blotting Secondary Antibodies table) diluted to 1:100-1:1000 in blocking buffer. Optimal antibody concentration is determined by titration. Incubate 1 hour at room temperature.
� Remove reagent by flicking. Wash three times with PBS containing 0.05% Tween-20 and slap plate dry, making sure the bottoms of the plates are dry.
� Wash wells once with diethanol-amine buffer (10 mM diethanolamine, 0.5 mM MgCl₂, pH 9.5) and slap plate dry.
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 �l/well. Allow to develop for 10-20 minutes or until positive control reaches an OD 405/490 of about 1.0. Stop reaction by adding 50 �l of 0.1 M EDTA, pH 7.5. Read plates on microtiter plate reader at OD 405/490.

8. Peptide Neutralization

� Blocking peptides are available as controls for all Antagene Inc�s affinity-purified rabbit and goat polyclonal antibodies raised against peptide antigens, and for some of our monoclonal antibodies. Antibody binding to antigen may be neutralized 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 �g/ml but is positive at a dilution of 50 ng/ml.
� For neutralization, combine antibody (at a concentration determined by the above method) with a five-fold (by weight) excess of blocking peptide in a small volume of PBS. Incubate for 2 hours at room temperature or overnight at 4� C.
� Following neutralization, dilute antibody/peptide mixture into appropriate working solution and proceed with the specific protocol for the desired test.

9. General Solutions

Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 8.0; 150 mM NaCl.

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.

Electrophoresis sample buffer (2x): Mix 1.0 ml glycerol, 0.5 ml -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 -mercaptoethanol and store at room temperature. Add -mercaptoethanol just before using.

Diaminobenzidine tetrahydrochloride (DAB): Dissolve 5 mg DAB (Sigma Chemicals) in 100 ml 100 mM Tris-HCl, pH 7.6, and add 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB solution daily.

RIPA buffer: 1x PBS, 1% Nonidet P-40 (Amaresco) or Igepal CA-630 (Sigma Chemicals), 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add inhibitors at time of use from the following stock solutions: 1) 10 mg/ml PMSF in isopropanol (add at 10 �l/ml RIPA) 2) Aprotinin (Sigma cat # A6279, available as liquid, add at 30 �l/ml RIPA) 3) 100 mM sodium orthovanadate in frozen aliquots (add at 10 �l/ml RIPA)

Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled H₂O.