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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.
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: 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 (weight to volume [w/v])
6. Blocking Buffer: 1X TBS, 0.1% Tween-20 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. While stirring, add 0.15 ml Tween-20 (100%).
7. Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T)
8. Bovine Serum Albumin (BSA)
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. Broad range protein marker,
11. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes or PVDF membranes.

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 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: EnoGene recommends loading prestained molecular weight markers to verify electrotransfer and determine molecular weights.
8. Electrotransfer to nitrocellulose or PVDF 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 HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in 10 ml of blocking 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. Incubate membrane with 10 ml HRP substrate solution with gentle agitation for 1 minute at room temperature.
NOTE: Substrate solution can be further diluted if signal response is too fast.
2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10-second exposure should indicate the proper exposure time.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following substrate solution incubation and declines over the following 2 hours.

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