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PE Mouse anti-CrkL (pY207)
PE Mouse anti-CrkL (pY207)

Analysis of CrkL (pY207) in human chronic myelogenous leukemia cell line. K562 cells (ATCC CCL-243) were either treated with the tyrosine kinase inhibitor, imatinib, at 25 μM (LC Laboratories, shaded histogram) for 2 hours at 37°C or untreated (open histogram, left figure). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-CrkL (pY207, Cat. No. 560788). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.

The specificity of mAb K30-391.50.80 was confirmed by western blot (right figure) using unconjugated antibody on lysates from control (lane 1) and imatinib-treated (lane 2) K562 cells. CrkL (pY207) is identified as a band of 39 kDa that has decreased intensity in the treated cells.

Analysis of CrkL (pY207) in human chronic myelogenous leukemia cell line. K562 cells (ATCC CCL-243) were either treated with the tyrosine kinase inhibitor, imatinib, at 25 μM (LC Laboratories, shaded histogram) for 2 hours at 37°C or untreated (open histogram, left figure). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-CrkL (pY207, Cat. No. 560788). Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.

The specificity of mAb K30-391.50.80 was confirmed by western blot (right figure) using unconjugated antibody on lysates from control (lane 1) and imatinib-treated (lane 2) K562 cells. CrkL (pY207) is identified as a band of 39 kDa that has decreased intensity in the treated cells.

Product Details
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BD Phosflow™
Human (QC Testing), Mouse, Rat (Predicted)
Mouse BALB/c IgG2a, κ
Phosphorylated Human, Mouse, and Rat CrkL Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1937309
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

This PE-conjugated antibody is suitable for intracellular staining of cell lines using BD Cytofix™ Fixation Buffer and BD Phosflow™ Perm Buffer III or IV. We have confirmed that the Alexa Fluor® 647 conjugate (Cat. No. 560790) is suitable for staining leukocytes from human peripheral blood and bone marrow.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560788 Rev. 2
Antibody Details
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K30-391.50.80

Crk-Like (CrkL) is an adaptor protein that is preferentially expressed in hematopoietic cells and is encoded by a gene that is homologous to the viral oncogene v-crk (chicken tumor virus no. 10 regulator of kinase). Its SH2 and SH3 domains bind to a variety of effector proteins, such as paxillin, p130Cas, c-Cbl, c-Abl, and C3G. These interactions are involved in the regulation of cellular migration, adhesion, and transformation. Tyrosine 207 (Y207) of CrkL is phosphorylated in hematopoietic cells that express the BCR-ABL fusion protein. This site may be a negative regulator of protein complex formation and biological activity.

The K30-391.50.80 monoclonal antibody recognizes the phosphorylated Y207 of human CrkL.

560788 Rev. 2
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560788 Rev.2
Citations & References
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Development References (3)

  1. Arai A, Nosaka Y, Kohsaka H, Miyasaka N, Miura O. CrkL activates integrin-mediated hematopoietic cell adhesion through the guanine nucleotide exchange factor C3G. Blood. 1999; 93(11):3713-3722. (Biology). View Reference
  2. Feller SM. Crk family adaptors-signalling complex formation and biological roles. Oncogene. 2001; 20:6348-6371. (Biology). View Reference
  3. Senechal K, Heaney C, Druker B, Sawyers CL. Structural requirements for function of the Crkl adapter protein in fibroblasts and hematopoietic cells. Mol Cell Biol. 1998; 18(9):5082-5090. (Biology). View Reference
560788 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.