Flow cytometric analysis of HeLa cells (blue) and RH30 cells (green) using SHMT2 (E7X5B) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
|Flow Cytometry||1:50 - 1:200|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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: 404
SHMT2 (E7X5B) Rabbit mAb recognizes endogenous levels of total SHMT2 protein. This antibody does not cross-react with SHMT1 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu402 of human SHMT2 protein.
Serine hydroxymethyltransferases 1 and 2 (SHMT1, SHMT2) are cytoplasmic and mitochondrial serine hydroxymethyltransferases, respectively (1,2). They catalyze the conversion of serine to glycine with the transfer of β-carbon from serine to tetrahydrofolate (THF) to form 5, 10-methylene-THF (1, 2). Research studies indicate that SHMT1 hemizygosity is associated with higher risk of intestinal cancer in mice of a certain genetic background (3). Suppression of SHMT2 was shown to block cell proliferation (4).
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XP is a registered trademark of Cell Signaling Technology, Inc.
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