Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In Milk)

For western blots, incubate membrane with diluted antibody in 5% w/v nonfat dry milk, 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. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
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): (#9997) To prepare 1L 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) (weight to volume [w/v]).
6. Blocking Buffer: 1X TBS with 5% w/v nonfat dry milk; for 150 ml, add 15 ml 10X TBS to 135 ml dH₂O, mix. Add 7.5 g nonfat dry milk and mix well.
7. Wash Buffer: (#9997) 1X TBS, 0.1% Tween-20 (TBS/T).
8. 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%).
9. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
10. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes (recommended).

B. Protein Blotting

A general protocol for sample preparation.

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 complete cell lysis and shear DNA (to 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: Loading of prestained molecular weight markers (#7720, 10 µl/lane) is recommended 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 as recommended in the product datasheet) in 10 ml 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 antibody dilution 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 membrane using an appropriate fluorescent scanner following the manufacturer’s recommendations.