Western blot analysis of extracts from HeLa cells using β-Actin (8H10D10) Mouse mAb #3700 as the primary antibody. Rabbit Anti-mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (left) and Rabbit Anti-mouse IgG (HRP Conjugate) #7076 (right) secondary antibodies were used.Learn more about how we get our images.
Western blot analysis of decreasing concentrations of total mouse IgG reduced and denatured in 1x SDS loading buffer with DTT using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (left) or Anti-Mouse IgG (HRP Conjugate) #7076 (right).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
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 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 19
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.
This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups.
Rabbit Anti-mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) recognizes native mouse IgG. It also recognizes the denatured and reduced mouse IgG light chains (about 25 kDa) on a western blot.
Monoclonal antibody is produced by immunizing animals with native total mouse IgG.
Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) only reacts with native IgG and denatured and reduced mouse IgG light chain. When performing immunopreciptiation (IP) followed by western blotting, the denatured mouse IgG heavy chains of the primary antibody used for IP run at approximately 50 kDa on the subsequent western blot and can often obscure bands of proteins that have similar molecular weights. Using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) as a secondary antibody will eliminate this problem.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.