Immunoblotting (Western Blotting)

Reagents and Equipment
Procedure
Troubleshooting Guide
References

The electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) to sheets of nitrocellulose was initially referred to as "Western" blotting. In order to avoid this geographical jargon, the term immunoblotting is used.

Reagents and Equipment

1. 0.3 M TRIZMA^® base (Product No. T1503), 20% methanol.
2. 0.025 M TRIZMA^® base (Product No. M1770), 20% methanol.
3. 0.02 M TRIZMA^® base, 20% methanol, 5.25 mg/ml epsilon-amino caproic acid (Product No. A2504).
4. Tris buffered saline (TBS) (Product No. T6664).
5. Tris buffered saline with 0.05% Tween 20 (TBS/Tween) (Product No. T9039).
6. Bovine serum albumin (BSA) (Product No. A9647).
7. Nitrocellulose (NC) membrane 0.45 5m pore size. 0.2 5m pore size nitrocellulose membrane may be used for low molecular weight molecules.
8. Blotting paper, extra thick.
9. Primary antibody.
10. Alkaline phosphatase-conjugated secondary antibody.
11. Phosphate buffered saline with 0.05% Tween 20 (PBS/Tween) (Product No. P3563).
12. SIGMA FAST™ BCIP/NBT Alkaline Phosphatase Substrate Tablets (Product No. B5655 or B1911 Liquid BCIP/NBT).
13. Minigel system (Product No. B2157).
14. 2000 volt power supply.
15. Semi-dry blotter (Product No. B2529).
16. Orbital shaker (Product No. Z367605).
17. Troughs 2-3 mm depth, 7-10 mm wide.

Procedure

SDS-PAGE
Protein Blotting
Immuno-Detection

SDS-PAGE

1. Carry out SDS-PAGE (cassette size approx. 80 x 80 mm). For analytical runs, load 5-20 micrograms of protein per well; for preparatory purposes, use 600-800 micrograms of total cell or tissue homogenate per gel. Apply constant voltage at 70-140 V or 1-2 hours until the tracking dye migrates 1.0 cm from the gel end. Many simple techniques for preparing gel slabs have been described in the literature and in the book: ''Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis'' by H.R. Maurer (de Gruyter; Berlin, New York, 1971).

Protein Blotting

1. Build the transfer "sandwich" onto the anode(+) plate as follows: 2 sheets blotting paper soaked in 0.3 M TRIZMA base, 20% methanol, 1 sheet blotting paper soaked in 0.025 M TRIZMA base, 20% methanol, NC membrane pre-wet with deionized water, Slab gel, 3 sheets blotting paper soaked with 0.02 M TRIZMA^. base, 20% methanol, 5.25 mg/ml epsilon-aminocaproic acid.
2. Carry out the transfer at 90 mA for 1.5 hours at room temperature.
3. Remove the NC membrane from the apparatus and air dry the NC blot thoroughly.

Note: The dry membrane may be stored at 2-8 °C between two sheets of blotting paper in a plastic sleeve.

Immuno-Detection

The following incubation and washing steps are carried out at room temperature on an orbital shaker platform. Primary antibody is diluted in 1% normal serum from the secondary antibody host animal in TBS. Other non-interfering proteins (e.g., BSA, hemoglobin, ovalbumin) may be substituted.

1. Cut the membrane into strips. Place the NC strips, with the side in contact with the gel during the transfer facing up, in the troughs.
2. Block NC blot using 5% w/v BSA in TBS, overnight at 4 °C or 2 hrs. at room temperature. The choice of blocking reagent depends on the type of probe that will be subsequently used in the overlay procedure and should be chosen accordingly.
3. Remove the blocking buffer.
4. Overlay the blot with 5 ml primary antibody at an appropriate dilution (generally 1:50-1:500). Incubate for 1-3 hours at room temperature on shaker.
5. Wash the NC strips four times for 5 minutes each, with sufficient TBS/Tween.
6. Incubate the strips for 1 hour in alkaline phosphatase-secondary antibody conjugate at an appropriate dilution. (1:30,000 for upgraded alkaline phosphatase conjugates) in TBS/Tween.
7. Wash the strips as in step 4. Rinse 3 times in TBS for 5 minutes each.
8. Prepare SIGMA FASTTM BCIP/NBT Tablets according to package directions. Liquid BCIP/NBT substrate may be used (Product No. B1911).
9. Incubate the NC strips in the substrate mixture for 10-30 minutes until color development.
10. Stop the reaction by washing the strips in several changes of distilled water.
11. Air dry the strips and store in the dark in a plastic sleeve.

Note: The dry membrane may be stored at 2-8 °C between two sheets of blotting paper in a plastic sleeve.

References

1. Burnette, W.N., Anal. Biochem., 112, 195-203 (1981).
2. Hames, B.D., and Rickwood, D. (eds.), "Gel Electrophoresis of Proteins: A Practical Approach", IRL Press Ltd., Oxford (1981).
3. Gershoni, J.M., and Palade, G.E., Anal. Biochem., 131, 1-15 (1983).

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