Western blot analysis of extracts from 293T cells, mock-transfected (-) or transfected with a construct expressing Cre recombinase (+), using Cre Recombinase (D7L7L) XP® Rabbit mAb (HRP Conjugate).
This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cre Recombinase (D7L7L) Rabbit mAb #15036.
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 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.
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: 11
Cre Recombinase (D7L7L) Rabbit mAb (HRP Conjugate) recognizes transfected and transgenic levels of total Cre recombinase protein.Species Reactivity:
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of bacteriophage-P1 Cre recombinase protein.
Cre recombinase is a bacteriophage-P1 enzyme required for maintenance of the phage genome as a monomeric plasmid in the lysogenic state (1,2). This enzyme mediates a site-specific recombination between two 34-base pair loxP sites. This reaction can be carried out in vitro, indicating that it does not require accessory factors (3). The Cre/Lox system has been used for a number of in vitro and in vivo applications including targeted gene deletions (4) and gene-specific humanized animal models (5). Resolution of the crystal structure of the Cre-Lox complex revealed that two Cre molecules interact with a single Lox site (6).
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