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82922
CD45R/B220 (RA3-6B2) Rat mAb (PE-Cy7® Conjugate)
Antibody Conjugates

Monoclonal Antibody - CD45R/B220 (RA3-6B2) Rat mAb (PE-Cy7® Conjugate), UniProt ID P06800, Entrez ID 19264 #82922

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Flow cytometric analysis of live mouse splenocytes using CD45R/B220 (RA3-6B2) Rat mAb (PE-Cy7® Conjugate) (solid line) compared to concentration-matched Rat Isotype Control (PE-Cy7® Conjugate) (dashed line).

To Purchase # 82922S

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rat IgG2a, kappa

Product Description

This Cell Signaling Technology antibody is conjugated to PE-Cy7® and tested in-house for direct flow cytometry analysis in mouse cells.

Product Usage Information

For optimal flow cytometry results, we recommend 0.125 μg of antibody per test.

Application Dilutions
Flow Cytometry 1:160

Storage:

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. This product is stable for 6 months when stored at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Immunostaining

  1. Count cells using a hemacytometer or alternative method.
  2. Aliquot 0.5-1x106 cells into each assay tube (by volume).
  3. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  4. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  5. Incubate for 1 hr at room temperature.
  6. Wash by centrifugation in 2-3 ml incubation buffer.
  7. Resuspend cells in the appropriate volume of PBS and analyze on flow cytometer.

posted June 2017

Protocol Id: 1504

Specificity / Sensitivity

CD45R/B220 (RA3-6B2) Rat mAb (PE-Cy7®) recognizes endogenous levels of total CD45R/B220 protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Human, Mouse

Source / Purification

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.

Background

The protein phosphatase (PTP) receptor CD45 is a type I transmembrane protein comprised of a pair of intracellular tyrosine phosphatase domains and a variable extracellular domain generated by alternative splicing (1). The catalytic activity of CD45 is a function of the first phosphatase domain (D1) while the second phosphatase domain (D2) may interact with and stabilize the first domain, or recruit/bind substrates (2,3). CD45 interacts directly with antigen receptor complex proteins or activates Src family kinases involved in the regulation of T- and B-cell antigen receptor signaling (1). Specifically, CD45 dephosphorylates Src-family kinases Lck and Fyn at their conserved negative regulatory carboxy-terminal tyrosine residues and upregulates kinase activity. Conversely, studies indicate that CD45 can also inhibit Lck and Fyn by dephosphorylating their positive regulatory autophosphorylation site. CD45 appears to be both a positive and a negative regulator that conducts signals depending on specific stimuli and cell type (1). Human leukocytes including lymphocytes, eosinophils, monocytes, basophils, and neutrophils express CD45, while erythrocytes and platelets are negative for CD45 expression (4).

The RA3-6B2 clone is raised against an isoform of CD45 known as CD45R/B220. CD45R/B220 is widely used as a marker for B cells, T cell subsets, and NK cell subsets in both human and mouse (5).

  1. Huntington, N.D. and Tarlinton, D.M. (2004) Immunol Lett 94, 167-74.
  2. Felberg, J. and Johnson, P. (2000) Biochem Biophys Res Commun 271, 292-8.
  3. Kashio, N. et al. (1998) J Biol Chem 273, 33856-63.
  4. Wang, Y. and Johnson, P. (2005) J Biol Chem 280, 14318-24.
  5. Hermiston, M.L. et al. (2003) Annu Rev Immunol 21, 107-37.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

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