Flow cytometric analysis of A-204 cells (blue) or MOLT-4 cells (green) using ERG (A7L1G) 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 ERG (A7L1G) Rabbit mAb #97249.
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
ERG (A7L1G) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total ERG protein. Based on sequence identity, this antibody should detect isoforms ERG1, ERG2, and ERG3. This antibody does not cross-react with Fli1.
Guinea Pig, Hamster, Horse, Pig, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ERG protein.
ETS-related gene (ERG) is a member of the E-26 transformation-specific (ETS) family of sequence-specific DNA-binding transcription factors (1). ERG plays important and highly conserved roles in vertebrate development. Early in embryonic development, ERG is highly expressed in the embryonic mesoderm and endothelium, where it plays a critical role in the formation of the vascular system, urogenital tract and bone development (2,3). Later in embryonic development, ERG functions to regulate the pluripotency of hematopoietic stem cells, endothelial cell homeostasis and angiogenesis (2,4-7). ERG expression is not restricted to development. In adult mouse, ERG is normally expressed in endothelial tissues, including adrenal, cartilage, heart, spleen, lymphatic endothelial and eosinophil cells (8). However, deregulation of ERG activity, often resulting from chromosomal rearrangements, has been implicated and linked to poor prognosis in a number of different cancers. Chromosomal translocations generating EWS/ERG chimeric proteins comprised of the amino-terminal transactivation domain of Ewing’s sarcoma breakpoint region 1 (EWS) and the carboxy-terminal ETS domain of ERG have been identified in 5-10% of Ewing’s sarcoma, an aggressive bone and soft tissue tumor (9). Chromosomal translocations between ERG and TLS/FUS or ERG and ELF4 have been implicated in acute myeloid leukemia (10, 11). Over-expression of ERG, resulting from gene fusion with the androgen-driven promoter of the TMPRSS2 gene, has been identified as a key driver of metastasis and marker for poor prognosis in prostate cancer (12).
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