Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In BSA)

For Western blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight.

NOTE: Two-color Western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment.

Products available from Cell Signaling Technology (CST) are linked by their respective catalog numbers.

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS).
2. 1X SDS Sample Buffer: (#7722, #7723) 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.
3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
4. 10X Tris Buffered Saline (TBS): To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X).
5. Nonfat Dry Milk: (#9999).
6. Blocking Buffer: 1X TBS with 5% w/v nonfat dry milk; for 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk and mix well.
7. Wash Buffer: (#9997) 1X TBS, 0.1% Tween-20 (TBS/T).
8. Bovine Serum Albumin (BSA): (#9998).
9. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% BSA; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. While stirring, add 20 μl Tween-20 (100%).
10. Secondary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% nonfat dry milk; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g nonfat dry milk and mix well. While stirring, add 20 μl Tween-20 (100%).
11. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
12. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends.

B. Protein Blotting

A general protocol for sample preparation is described below.

1. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with cold 1X PBS; aspirate.
3. Lyse cells by adding 1X SDS sample buffer (100 μl per well of 6-well plate or 500 μl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
4. Sonicate for 10–15 seconds to shear DNA and reduce sample viscosity.
5. Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
6. Microcentrifuge for 5 minutes.
7. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: CST recommends loading prestained molecular weight markers (#7720, 10 μl/lane) to verify electrotransfer and to determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths.

8. Electrotransfer to nitrocellulose membrane.

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature.
2. Incubate membrane in 25 ml of blocking buffer for 1 hour at room temperature.
3. Wash three times for 5 minutes each with 15 ml of TBS/T.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
5. Wash three times for 5 minutes each with 15 ml of TBS/T.
6. Incubate membrane with fluorophore-conjugated secondary antibody (1:5000-1:25,000 dilution of 1 mg/ml stock) in 10 ml of secondary antibody incubation buffer with gentle agitation for 1 hour at room temperature.
7. Wash three times for 5 minutes each with 15 ml of TBS/T.

D. Detection of Proteins

1. Drain membrane of excess TBS/T and allow to dry.
2. Scan membane using an appropriate fluorescent scanner following the manufacturer's recommendations.