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.Learn more about how we get our images.
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
A general protocol for sample preparation is described below.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2009
revised May 2014
Protocol Id: 564
Affinity purified mouse anti-rabbit IgG (Conformation Specific) antibody. This product has been optimized for use as a secondary antibody in Western blotting applications.
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.
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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