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16606
ERG (A7L1G) Rabbit mAb (PE Conjugate)
Antibody Conjugates

ERG (A7L1G) Rabbit mAb (PE Conjugate) #16606

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Flow cytometric analysis of A-204 cells (blue) or MOLT-4 cells (green) using ERG (A7L1G) Rabbit mAb (PE Conjugate).

To Purchase # 16606S
Product # Size Price
16606S
100 µl  (50 tests) $ 305

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Description

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.

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage:

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

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.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

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.

B. Fixation

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.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

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.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology:

Rat, Hamster, Pig, Guinea Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ERG protein.

Background

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).

  1. Adamo, P. and Ladomery, M.R. (2016) Oncogene 35, 403-14.
  2. Birdsey, G.M. et al. (2008) Blood 111, 3498-506.
  3. Vijayaraj, P. et al. (2012) Development 139, 3973-85.
  4. Ng, A.P. et al. (2011) Blood 118, 2454-61.
  5. Birdsey, G.M. et al. (2015) Dev Cell 32, 82-96.
  6. Lathen, C. et al. (2014) Circulation 130, 1179-91.
  7. McLaughlin, F. et al. (2001) Blood 98, 3332-9.
  8. Mohamed, A.A. et al. (2010) J Cancer 1, 197-208.
  9. Chen, S. et al. (2016) Genes Chromosomes Cancer 55, 340-9.
  10. Ichikawa, H. et al. (1994) Cancer Res 54, 2865-8.
  11. Moore, S.D. et al. (2006) Leuk Res 30, 1037-42.
  12. Tomlins, S.A. et al. (2005) Science 310, 644-8.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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