Immunoprecipitation of PRAS40 from HeLa cells using PRAS40 (D23C7) XP® Rabbit mAb #2691 (lane 2). Lane 1 contains input control lysate. All lanes are probed with PRAS40 (D23C7) Rabbit mAb #2691 as the primary antibody. Secondary antibodies include Anti-rabbit IgG, HRP-linked Antibody #7074 (panel A) and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 (panel B).Learn more about how we get our images.
Immunoprecipitation of Beclin-1 from HeLa cells using Beclin-1 (D40C5) Rabbit mAb #3495 (lane 2). Lane 1 contains input control lysate. All lanes are probed with Beclin-1 (D40C5) Rabbit mAb #3495 as the primary antibody. Secondary antibodies include Anti-rabbit IgG, HRP-linked Antibody #7074 (panel A) and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 (panel B).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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 October 2016
Protocol Id: 10
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 antibodies.
Affinity purified mouse anti-rabbit IgG (Conformation Specific) HRP Conjugate antibody.
Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) 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.
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