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83090
CD45 (30-F11) Rat mAb (violetFluor™ 450 Conjugate)

CD45 (30-F11) Rat mAb (violetFluor™ 450 Conjugate) #83090

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rat IgG2b, kappa
Flow Cytometry

Flow cytometric analysis of live mouse splenocytes using CD45 (30-F11) Rat mAb (violetFluor™ 450 Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (violetFluor™ 450 Conjugate) #52572 (dashed line).

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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

For optimal flow cytometry results, we recommend 0.5μg of antibody per test.

Application Dilutions
Flow Cytometry 1:40
Storage:

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. This product is stable for 6 months when stored at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.

CD45 (30-F11) Rat mAb (violetFluor™ 450 Conjugate) recognizes endogenous levels of total mouse CD45 protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Mouse

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.

This Cell Signaling Technology antibody is conjugated to violetFluor™ 450 and tested in-house for direct flow cytometric analysis in mouse cells.

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 30-F11 antibody is widely used as a leukocyte marker for B cells, T cell subsets, and NK cell subsets.

  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.
Entrez-Gene Id
19264
Swiss-Prot Acc.
P06800
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
violetFluor is a registered trademark of Tonbo Biosciences.

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To Purchase # 83090S

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Product # Size Price
83090S
100 µg $ 149.0