Mouse anti-Human CD45 Antibody | anti-CD45 antibody
Mouse anti CD45
The sensitivity of VIT200 mAb is determined by staining well-definted blood samples from representative donors with serial-fold mAb dilutions to obtain a titration curve that allows relating the mAb concentration to the percentage of stained cells and geometric MFI (mean fluorescence intensity). For this purpose, a mAb-concentration range is selected to include both the saturation point (i.e. the mAb dilution expected to bind all epitopes on the target cell) and the detection threshold (i.e. the mAb dilution expected to represent the least amount of mAb needed to detect an identical percentage of cells). In practice, 50 ul of leukocytes containing 10^7 cells/ml are stained with 20 ul mAb of various dilutions to obtain a titration curve and to identify the saturation point and detection threshold. The final concentration of the product is then adjusted to be at least 3-fold above the detection threshold. In addition and to control lot-to-lot variation, the given lot is compared and adjusted to fluorescence standards with defined intensity.
Proposed staining procedure for whole blood in short:
- For each sample add 50 ul of EDTA anti-coagulated blood to a 3-5 ml tube
- Add 20 ul of the appropriate monoclonal antibody conjugate
- Incubate the tube for 15 minutes at 4 degree C or at room temperature in the dark
- Add 100 ul NM-LYSE to each tube and incubate for 10 minutes at room temperature
- Add 3-4 ml of destilled water and vortex, incubate for 5-10 minutes at room temperature
- Centrifuge tube for 5 minutes at 300 g
- Aspirate supernatant and resuspend pellet in 0.3 ml of sheath fluid
- Analyze immediately or store samples at 2-8 degree C in the dark and analyze within 24 hours
Proposed staining procedure for MNC in short:
- Carefully add 20 ul antibody conjugate and 50-100 ul MNC to the bottom of a tube
- Vortex at low speed for 1-2 seconds
- Incubate for 15-30 minutes at 2-8 degree C or at room temperature
- Centrifuge tubes for 5 minutes at 300 g
- Remove supernatant, resuspend cells in 2-5 ml of phosphate buffered saline (PBS) and centrifuge cells again for 5 minutes at 300 g
- Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml 1 % formaldehyde and store them at 2-8 degree C in the dark. Analyze fixed cells within 24 hours
Indirect Immunofluorescence (Staining Procedure)
- Mix 20 ul purified antibody with 50 ul whole blood or MNC suspension
- Incubate for 15 minutes at 2-8 degree C
- Wash cells with phosphate buffered saline (PBS)
- Add to cell pellet 20 ul of affinity purified, fluorochrome labeled F(ab')2 anti mouse Ig antibodies
- Incubate for 15 minutes at 2-8 degree C
- Wash cells with phosphate buffered saline (PBS) or proceed as described for direct staining
The VIT200 antibody permits the identification and enumeration of human leukocytes using flow cytometry. Results must be put within the context of other tests as well as the clinical history of the patient by a certified professional before final interpretation. Analysis performed with this antibody should be paralleled by positive and negative controls.
Brocklebank, A. M. & Sparrow, R. L. (2001) Cytometry 46, 254-61.
Cobbold, S., Hale, G. & Waldmann, H. (1987) In Leukocyte Typing III (Oxford University Press, Oxford-New York-Tokyo) p788-803.
Dalchau, R., Kirkley, J. & Fabre, J. W. (1980) Eur J Immunol 10, 737-44.
Nicholson, J. K., Hubbard, M. & Jones, B. M. (1996) Cytometry 26, 16-21.
Omary, M. B., Trowbridge, I. S. & Battifora, H. A. (1980) J Exp Med 152, 842-52
Schraven, B., Roux, M., Hutmacher, B. & Meuer, S. C. (1989) Eur J Immunol 19, 397-403.
Sugita, K., Majdic, O., Stockinger, H., Holter, W., Burger, R. & Knapp, W. (1987) Transplantation 43, 570-4.
Sun, T., Sangaline, R., Ryder, J., Gibbens, K., Rollo, C., Stewart, S. & Rajagopalan, C. (1997) Am J Clin Pathol 108, 152-7.
Thomas, M. L. (1989) Annu Rev Immunol 7, 339-69.
NCBI and Uniprot Product Information
NCBI Description
The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitosis, and oncogenic transformation. This PTP contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains, and thus is classified as a receptor type PTP. This PTP has been shown to be an essential regulator of T- and B-cell antigen receptor signaling. It functions through either direct interaction with components of the antigen receptor complexes, or by activating various Src family kinases required for the antigen receptor signaling. This PTP also suppresses JAK kinases, and thus functions as a regulator of cytokine receptor signaling. Alternatively spliced transcripts variants of this gene, which encode distinct isoforms, have been reported. [provided by RefSeq, Jun 2012]
Uniprot Description
CD45: a receptor type protein tyrosine phosphatase protein. Contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains. The first PTPAse domain has enzymatic activity, while the second one seems to affect the substrate specificity of the first one. Specifically expressed in hematopoietic cells. An essential regulator of T- and B-cell antigen receptor signaling. Functions through either direct interaction with components of the antigen receptor complexes, or by activating various Src family kinases required for the antigen receptor signaling. Suppresses JAK kinases, and thus functions as a regulator of cytokine receptor signaling. Four alternatively spliced isoforms have been described.
Protein type: Membrane protein, integral; EC 3.1.3.48; Motility/polarity/chemotaxis; Receptor protein phosphatase, tyrosine
Chromosomal Location of Human Ortholog: 1q31-q32
Cellular Component: focal adhesion; membrane; integral to plasma membrane; plasma membrane; lipid raft; external side of plasma membrane
Molecular Function: protein binding; transmembrane receptor protein tyrosine phosphatase activity; protein tyrosine phosphatase activity; protein kinase binding
Biological Process: B cell proliferation; axon guidance; substrate-bound cell migration, cell release from substrate; regulation of cell cycle; protein amino acid dephosphorylation; positive regulation of antigen receptor-mediated signaling pathway; T cell receptor signaling pathway; bone marrow development; B cell receptor signaling pathway; stem cell development; cell surface receptor linked signal transduction; dephosphorylation; release of sequestered calcium ion into cytosol; positive regulation of protein kinase activity; negative regulation of T cell mediated cytotoxicity; positive regulation of B cell proliferation; negative regulation of protein kinase activity; hemopoietic progenitor cell differentiation; positive regulation of T cell proliferation; negative regulation of cytokine and chemokine mediated signaling pathway; immunoglobulin biosynthetic process; defense response to virus; T cell differentiation
Disease: Hepatitis C Virus, Susceptibility To; Severe Combined Immunodeficiency, Autosomal Recessive, T Cell-negative, B Cell-positive, Nk Cell-positive
Research Articles on CD45
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Product Notes
The CD45 ptprc (Catalog #AAA570368) is an Antibody produced from Mouse and is intended for research purposes only. The product is available for immediate purchase. The Mouse anti CD45 reacts with Human and may cross-react with other species as described in the data sheet. AAA Biotech's CD45 can be used in a range of immunoassay formats including, but not limited to, Direct Immunofluorescence (IF), Indirect Immunofluorescence (IF), Flow Cytometry (FC/FACS). Direct Immunofluorescence (Staining Procedure) Fluorochrome labeled antibodies are designed for use with either whole blood or isolated mononuclear cell (MNC) preparations. Proposed staining procedure for whole blood in short: - For each sample add 50 ul of EDTA anti-coagulated blood to a 3-5 ml tube - Add 20 ul of the appropriate monoclonal antibody conjugate - Incubate the tube for 15 minutes at 4 degree C or at room temperature in the dark - Add 100 ul NM-LYSE to each tube and incubate for 10 minutes at room temperature - Add 3-4 ml of destilled water and vortex, incubate for 5-10 minutes at room temperature - Centrifuge tube for 5 minutes at 300 g - Aspirate supernatant and resuspend pellet in 0.3 ml of sheath fluid - Analyze immediately or store samples at 2-8 degree C in the dark and analyze within 24 hours Proposed staining procedure for MNC in short: - Carefully add 20 ul antibody conjugate and 50-100 ul MNC to the bottom of a tube - Vortex at low speed for 1-2 seconds - Incubate for 15-30 minutes at 2-8 degree C or at room temperature - Centrifuge tubes for 5 minutes at 300 g - Remove supernatant, resuspend cells in 2-5 ml of phosphate buffered saline (PBS) and centrifuge cells again for 5 minutes at 300 g - Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml 1 % formaldehyde and store them at 2-8 degree C in the dark. Analyze fixed cells within 24 hours Indirect Immunofluorescence (Staining Procedure) - Mix 20 ul purified antibody with 50 ul whole blood or MNC suspension - Incubate for 15 minutes at 2-8 degree C - Wash cells with phosphate buffered saline (PBS) - Add to cell pellet 20 ul of affinity purified, fluorochrome labeled F(ab')2 anti mouse Ig antibodies - Incubate for 15 minutes at 2-8 degree C - Wash cells with phosphate buffered saline (PBS) or proceed as described for direct staining. Researchers should empirically determine the suitability of the CD45 ptprc for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "CD45, Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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