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Western Blotting

Western blot analysis of decreasing concentrations of total rabbit IgG, reduced and denatured in 1X SDS loading buffer with DTT, using Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (Panel A), Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 (Panel B), or Anti-rabbit IgG, HRP-linked Antibody #7074 (Panel C). For Panels A and B, the bound mouse anti-rabbit IgG mAb was detected using Anti-mouse IgG, HRP-linked Antibody #7076. The positions of the reduced and denatured rabbit IgG heavy and light chains are indicated.

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Western Immunoblotting Protocol (for Conformation Specific 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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. Nonfat Dry Milk: (#9999).
  8. 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.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. 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.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb: (#3678).
  16. Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
  17. Detection Reagent: SignalFire™ ECL Reagent (#6883).

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 (Conformation Specific) (L27A9) mAb (#3678) (1:2000) in 10 ml 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. 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.
  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

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. 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.
  3. 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 May 2014

protocol id: XXX

Product Usage Information

Storage: -20°C

Product Description

Affinity purified mouse anti-rabbit IgG (Conformation Specific) antibody. This product has been optimized for use as a secondary antibody in Western blotting applications.


Specificity / Sensitivity

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb recognizes the native rabbit IgG. It does not recognize the denatured and reduced rabbit IgG heavy (about 50 kDa) or light (about 25 kDa) chains on western blot.


Source / Purification

Monoclonal antibody is produced by immunizing animals with native total rabbit IgG.

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb only reacts with native IgG and does not bind to the denatured and reduced rabbit IgG heavy chain or light chain. When performing immunopreciptiation (IP) followed by western blotting, the denatured rabbit IgG light and heavy chains of the primary antibody used for IP run at approximately 25 and 50 kD, respectively, on the subsequent western blot and can often obscure bands of proteins that have similar molecular weights. Using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb as a secondary antibody will eliminate this problem.



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