Protocols

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Western Blotting with Horseradish Peroxidase Conjugates or Alkaline Phosphatase Conjugates

SAMPLE PREPARATION

For Protein Concentration Determination of Cell Culture

1. Decant medium from 10 cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS).
2. Aspirate excess PBS.
3. Add 1 ml boiling lysis buffer (1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris pH 7.4).
4. Scrape cells from dish, transfer to a microcentrifuge tube, and boil for 5 minutes in a boiling water bath. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle.
5. Centrifuge the sample for 5 minutes to pellet insoluble material, and collect the supernatant, which contains the cell lysate.
6. Dilute an aliquot of the cell lysate sample at least 10-fold for the BCA (Pierce) protein concentration assay (SDS concentration must be below 0.1% to avoid interference with the colorimetric reading).

For Protein Gel Electrophoresis of Cell Culture (without determining protein concentration)

1. Decant medium from 10 cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS).
2. Aspirate excess PBS.
3. Add 1 ml boiling 2X concentrated electrophoresis sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% ß-mercaptoethanol).
4. Scrape cells from dish, transfer to a microcentrifuge tube, and boil for an additional 5 minutes. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle. Centrifuge the sample for 10 minutes to pellet insoluble material, and collect the supernatant (cell lysate).
5. The cell lysate sample is now ready for loading onto your gel.

For Protein Concentration Determination of Whole Tissue

1. Rapidly homogenize every 0.25 g tissue in 3.5 ml of boiling lysis buffer (1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris pH 7.4).
2. Microwave for 10–15 seconds.
3. Centrifuge the homogenate (16,000 x g, 15°C) for 5 minutes to pellet insoluble material, then discard pellet.
4. Dilute an aliquot of the tissue lysate sample at least 10-fold for the BCA (Pierce) protein concentration assay.

POLYACRYLAMIDE GEL ELECTROPHORESIS

Guidelines for choosing the percent gel to be used for certain molecular weight proteins (based on 37:1 acrylamide: bis acrylamide ratio)

4-5% gels: > 250 kDa

7.5% gels: 250-120 kDa

10% gels: 120-40 kDa

13% gels: 40-15 kDa

15% gels: < 20 kDa

Gel Electrophoresis

1. If the cell lysate is not already in electrophoresis sample buffer, add an equal volume of 2X sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% ß-mercaptoethanol) to all samples and boil for 3–5 minutes.
2. Apply 5-20 µg total protein of cell or tissue lysate to each well of a 0.75–1.0 mm thick gel. For thicker gels (1.5 mm thick), apply up to 25–40 µg in each well. Please refer to the antibody datasheet for the appropriate positive control cell lysate.
3. Electrophorese until the bromophenol blue in the samples reaches the bottom of the gel. Turn off power supply. Keep gels in running buffer until ready to transfer.

PROTEIN BLOTTING

Wet Transfer

Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH, pH adjusted to 8.0.

Note: Since extra negative charges are needed to reach 1 Amp in a wet transfer system, adjust the pH of the transfer buffer to approximately pH 8.0 using NaOH.

1. For transfer of proteins smaller than 20 kDa, transfer proteins from gel to PVDF (polyvinylidene difluoride) membrane at 1Amp constant current for 45 mins or equivalent (250 mAmp for 3 hours or 500 mAmp for 90 minutes) in transfer buffer.
2. For transfer of proteins smaller than 120 kDa, transfer proteins from gel to PVDF membrane at 1 Amp constant current for 1 hour or equivalent (250 mAmp for 4 hours or 500 mAmp for 2 hours) in transfer buffer.
3. For proteins larger than 120 kDa, transfer to PVDF membrane at 1 Amp constant current for 90 minutes or equivalent (250 mAmp for 6 hours or 500 mAmp for 3 hours) in transfer buffer + SDS.
4. For Proteins larger than 250 kDa, transfer to PVDF membrane at 1 Amp constant current for 1 hour and 45 minutes or equivalent (500 mAmp for 3.5 hours) in transfer buffer + SDS.

* To ensure complete transfer of large molecular weight proteins (as in 3 and 4 above), 0.05% SDS can be added to the transfer buffer.

Semi-Dry Transfer

For transfer of proteins from 10% or 13% gels to PVDF membranes semi-dry transfer can also be used. Transfer proteins to PVDF membrane at 1.2 mAmp/cm² for 1 hour and 45 minutes in transfer buffer.

Optional

If blots are not to be used for colorimetric detection, visualize the transferred proteins by staining the membrane for 15 minutes with India ink (Higgins black India ink, Eberhard Faber) diluted 1:1000 in wash buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20). Rinse excess stain with wash buffer before blocking.

Ponceau S can be used if the blot is to be used for colorimetric assay as it is a reversible dye. Stain with 0.1% Ponceau S for 5 minutes and then visualize protein bands. To remove, rinse with distilled water and then immerse in an aqueous solution of 0.1 M NaOH until bands disappear (10 to 30 seconds), and then rinse the membrane with distilled water before blocking.

BLOCKING

FOR ALL ANTIBODIES EXCEPT PHOSPHOTYROSINE

1. Remove the blot from the transfer apparatus or staining tray and immediately place into blocking buffer (5% non-fat dry milk, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20).
2. Incubate the blot for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.

FOR PHOSPHOTYROSINE ANTIBODIES

1. Remove the blot from the transfer apparatus or staining tray and immediately place into blocking buffer (1% BSA, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20).
2. Incubate the blot for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.

INCUBATION WITH HORSERADISH PEROXIDASE CONJUGATED ANTIBODY

NOTE: THE INCLUSION OF SODIUM AZIDE IS TO BE AVOIDED IN ALL STEPS USING HRPO (HORSERADISH PEROXIDASE) CONJUGATES.

1. Dilute HRPO conjugated antibody in the corresponding blocking buffer
2. Decant blocking buffer from the blot, add the diluted antibody solution and incubate with agitation for 30 minutes at 37°C, one hour at room temperature or overnight at 4°C.

   SUBSTRATE INCUBATION

3. Decant the horseradish conjugated antibody solution, add wash buffer (10 mM Tris pH 7.5, 100 mMNaCl, 0.1% Tween 20), and wash for 30 minutes with agitation, changing the wash buffer every 3-5 minutes.
4. Decant wash buffer and place the blot in a plastic bag or clean tray containing chemiluminescent working solution (0.125 ml/cm²). Rotate the bag or tray to allow the solution to cover the surface of the membrane for 1-5 minutes.
5. Remove blot from the bag or tray and place it between two pieces of write-on acetate transparency film. Smooth over covered blot to remove air bubbles and excess substrate.

   EXPOSE TO X-RAY FILM OR ANY SENSITIVE SCREEN. AN INITIAL EXPOSURE OF 10-60 SECONDS IS RECOMMENDED FOR FILM.

INCUBATION WITH ALKALINE PHOSPHATASE CONJUGATED ANTIBODIES

1. Dilute the AP conjugated antibody in the corresponding blocking buffer.
2. Decant the blocking buffer from the blot, add the antibody solution, and incubate with agitation for 30 minutes at 37°C, one hour at room temperature, or overnight at 4°C.

   PREPARATION OF CHROMAGEN REAGENTS

   Prepare BCIP (bromochloroindolyl phosphate) by dissolving 0.5 grams of the disodium salt in 10 ml of 100% dimethylformamide. Prepare NBT (nitro blue tetrazolium) in a glass vial by dissolving 0.5 grams in 10 ml 70% dimethylformamide. Store separately at 4°C in dark containers.