Immunoprecipitation of β-catenin from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423, and lane 3 is β-Catenin (D10A8) XP® Rabbit mAb (Sepharose® Bead Conjugate) #12475. Western blot analysis was performed using β-Catenin (D10A8) XP® Rabbit mAb #8480 as the primary antibody and Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) as the secondary antibody.
This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups.
Supplied in 136 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
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 August 2016
Protocol Id: 29
Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) recognizes the light chain of rabbit IgG, either in its native conformation or in its denatured and reduced form. It does not bind to the denatured and reduced rabbit IgG heavy chain (about 50 kDa) on western blot.
Monoclonal antibody is produced by immunizing animals with native total rabbit IgG.
Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) only reacts with the light chain of rabbit IgG and does not bind to the denatured and reduced rabbit IgG heavy chain. When performing immunoprecipitation (IP) followed by western blotting, the denatured rabbit IgG heavy chain of the primary antibody used for IP runs at approximately 50 kDa on the subsequent western blot and can often obscure bands of proteins that have a similar molecular weight. Using Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) as a secondary antibody will eliminate this problem.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Sepharose is a registered trademark of GE Healthcare.
Tween is a registered trademark of ICI Americas, Inc.