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.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 liter of 1X PBS, add 50 ml 20X PBS to 950 ml water, mix.
- 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.
- Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
- 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).
- Nonfat Dry Milk: (#9999) (weight to volume [w/v]).
- 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%).
- Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T).
- Bovine Serum Albumin (BSA): (#9998).
- 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%).
- 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).
- Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
- 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.
- Treat cells by adding fresh media containing regulator for desired time.
- Aspirate media from cultures; wash cells with 1X PBS; aspirate.
- 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.
- Sonicate for 10–15 seconds for complete cell lysis and to shear DNA (to reduce sample viscosity).
- Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
- Microcentrifuge for 5 minutes.
- 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.
- 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
- (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature.
- Incubate membrane in 25 ml of blocking buffer for one hour at room temperature.
- Wash three times for 5 minutes each with 15 ml of TBS/T.
II. Primary Antibody Incubation
- 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.
- Wash three times for 5 minutes each with 15 ml of TBS/T.
- 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.
- Wash three times for 5 minutes each with 15 ml of TBS/T.
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- 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.
- Wash three times for 5 minutes each with 15 ml of TBS/T.
- Proceed to detection step in section D.
D. Detection of Proteins
- 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.
- 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.