Flow cytometric analysis of Jurkat cells, untreated (green) or etoposide-treated (blue), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 647 Conjugate) #9720 compared to Rabbit IgG Isotype Control (Alexa Fluor® 647 Conjugate) (red).
Flow cytometric analysis of Jurkat cells, untreated (green) or LY294002- and wortmannin-treated (blue), using Phospho-Akt (Ser473) (193H12) Rabbit mAb (Alexa Fluor® 647 Conjugate) #2337 compared to Rabbit IgG Isotype Control (Alexa Fluor® 647 Conjugate) (red).
Rabbit IgG was conjugated to Alexa Fluor® 647 fluorescent dye under optimal conditions, and tested in-house for direct flow cytometric analysis of human and mouse cells. Alexa Fluor® dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugated of the Alexa Fluor® 647 dye produce far-red-fluorescence emission with a peak at 655 nm.
Note: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. See Directions on Use.
Important Note on Use: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. Higher background fluorescence may result if excessive amounts of rabbit IgG isotype control are used. Do not use this antibody at 1:50 dilution in the same way as recommended for other CST Alexa Fluor® conjugated antibodies.
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/flowdyes for a 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 August 2019
Protocol Id: 407
The purified antibody is not directed against any known antigen. It functions as an isotype control for rabbit antibodies.
The antibody was isolated from naive rabbit sera and purified by affinity chromotography. The antibody was conjugated to Alexa Fluor® 647 under optimal conditions with a F/P ratio of 2-6.
Isotype control antibodies are used to estimate the nonspecific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.