Western Immunoblotting Protocol (for Light-Chain Specific 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 reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
- 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
- 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
- 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
- 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
- Nonfat Dry Milk: (#9999).
- Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
- Wash Buffer: (#9997) 1X TBST.
- Bovine Serum Albumin (BSA): (#9998).
- Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
- Biotinylated Protein Ladder Detection Pack: (#7727).
- Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
- Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
- Mouse Anti-Rabbit IgG (Light-Chain Specific) mAb.
- Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
- Detection Reagent: SignalFire™ ECL Reagent (#6883).
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) mAb (1:2000) in 10 ml 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.
- 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.
- Wash three times for 5 minutes each with 15 ml of TBS/T.
- Proceed to detection step in section D.
D. Detection of Proteins
Directions for Use:
- Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
- Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
- Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
* Avoid repeated exposure to skin.
posted June 2009
revised August 2016
Protocol Id: 29