Flow cytometric analysis of 293T cells, untransfected (left peak) and transfected with a construct expressing Cre recombinase (right peak), using Cre Recombinase (D7L7L) XP® Rabbit mAb (PE Conjugate).
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cre Recombinase (D7L7L) Rabbit mAb #15036.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Cre Recombinase (D7L7L) XP® Rabbit mAb (PE Conjugate) recognizes transfected and transgenic levels of total Cre recombinase protein.
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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