Western Blotting Protocol (for Conformation and Light-Chain Specific and Anti-Biotin Secondary Antibodies)

For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet for recommended Primary Antibody Dilution Buffer and recommended antibody dilution.

A. Solutions and Reagents

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

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 liter of 1X PBS, add 50 ml 20X PBS to 950 ml water, 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 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) (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): (#9998).
9. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% BSA or 5% nonfat dry milk as indicated on primary antibody datasheet; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA or nonfat dry milk and mix well. While stirring, add 20 μl Tween-20 (100%).
10. Phototope^®-HRP Western Blot Detection System: (anti-rabbit #7071)(anti-mouse #7072) Includes biotinylated protein ladder (#7727), secondary antibody conjugated to horseradish peroxidase (HRP) (anti-rabbit #7074)(anti-mouse #7076), anti-biotin HRP-linked antibody (#7075), LumiGLO chemiluminescent reagent and peroxide (#7003).
11. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
12. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used.

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 for complete cell lysis and to 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: CST recommends loading prestained molecular weight markers (#7720, 10 μl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 μl/lane) to 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.

I. Membrane Blocking

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 one hour at room temperature.
3. Wash three times for 5 minutes each with 15 ml of TBS/T.

II. Primary Antibody Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 minutes each with 15 ml of TBS/T.
3. Incubate membrane with Mouse anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (1:2000) or Anti-Biotin (D5A7) Rabbit mAb (#5597) in 10ml of blocking buffer with gentle agitation for 1 hour at room temperature.
4. Wash three times for 5 minutes each with 15 ml of TBS/T.

5. For Mouse anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and
   Mouse anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678:

   Incubate membrane with Anti-mouse IgG, HRP-linked antibody (#7076) (1:2000) and Anti-biotin, HRP-linked Antibody (#7075) (1:1000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for one hour at room temperature.

   For Anti-Biotin (D5A7) Rabbit mAb #5597:

   Incubate membrane with Anti-Rabbit IgG, HRP-linked antibody (#7074) (1:2000) in 10 ml of blocking buffer with gentle agitation for one hour at room temperature.

6. Wash three times for 5 minutes each with 15 ml of TBS/T.
7. Proceed to detection step in section D.

D. Detection of Proteins

1. Incubate membrane with 10 ml LumiGLO (0.5 ml 20X LumiGLO, 0.5 ml 20X Peroxide and 9.0 ml Milli-Q water) with gentle agitation for one minute at room temperature. NOTE: LumiGLO substrate 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 LumiGLO incubation and declines over the following 2 hours.