Recombinant
RabMAb

Recombinant Anti-CD45 antibody [EP322Y] (ab40763)

Overview

  • Product name
    Anti-CD45 antibody [EP322Y]
    See all CD45 primary antibodies
  • Description
    Rabbit monoclonal [EP322Y] to CD45
  • Host species
    Rabbit
  • Specificity
    ab40763 recognizes cytoplasmic domain of CD45.
  • Tested applications
    Suitable for: WB, Flow Cyt, ICC/IF, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to residues in the C-terminal domain of human CD45.

  • Positive control
    • Flow Cyt : Jurkat cells. IHC-P: Human kidney, colon lymphoid, tonsil and spleen tissue. ICC/IF: Jurkat cells. WB: Jurkat, HuT-78 and Raji cell lysate.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab40763 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/5000. Detects a band of approximately 200 kDa (predicted molecular weight: 147 kDa).
Flow Cyt 1/15 - 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Permeabilization and intracellular staining is necessary.

ICC/IF 1/100.
IHC-P 1/250.
IHC-Fr Use at an assay dependent concentration.

Target

  • Function
    Protein tyrosine-protein phosphatase required for T-cell activation through the antigen receptor. Acts as a positive regulator of T-cell coactivation upon binding to DPP4. The first PTPase domain has enzymatic activity, while the second one seems to affect the substrate specificity of the first one. Upon T-cell activation, recruits and dephosphorylates SKAP1 and FYN.
  • Involvement in disease
    Defects in PTPRC are a cause of severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell-positive/NK-cell-positive (T(-)B(+)NK(+) SCID) [MIM:608971]. A form of severe combined immunodeficiency (SCID), a genetically and clinically heterogeneous group of rare congenital disorders characterized by impairment of both humoral and cell-mediated immunity, leukopenia, and low or absent antibody levels. Patients present in infancy recurrent, persistent infections by opportunistic organisms. The common characteristic of all types of SCID is absence of T-cell-mediated cellular immunity due to a defect in T-cell development.
    Genetic variations in PTPRC are involved in multiple sclerosis susceptibility (MS) [MIM:126200]. MS is a neurodegenerative disorder characterized by the gradual accumulation of focal plaques of demyelination particularly in the periventricular areas of the brain. Peripheral nerves are not affected. Onset usually in third or fourth decade with intermittent progression over an extended period. The cause is still uncertain.
  • Sequence similarities
    Belongs to the protein-tyrosine phosphatase family. Receptor class 1/6 subfamily.
    Contains 2 fibronectin type-III domains.
    Contains 2 tyrosine-protein phosphatase domains.
  • Domain
    The first PTPase domain interacts with SKAP1.
  • Post-translational
    modifications
    Heavily N- and O-glycosylated.
  • Cellular localization
    Membrane. Membrane raft. Colocalized with DPP4 in membrane rafts.
  • Information by UniProt
  • Database links
  • Alternative names
    • B220 antibody
    • CD 45 antibody
    • CD45 antibody
    • CD45 antigen antibody
    • CD45R antibody
    • GP180 antibody
    • L-CA antibody
    • LCA antibody
    • Leukocyte common antigen antibody
    • loc antibody
    • Ly-5 antibody
    • LY5 antibody
    • Ly5, homolog of antibody
    • Lyt-4 antibody
    • OTTHUMP00000033813 antibody
    • OTTHUMP00000033816 antibody
    • OTTHUMP00000033817 antibody
    • OTTHUMP00000038574 antibody
    • Protein tyrosine phosphatase receptor type c polypeptide antibody
    • Protein tyrosine phosphatase, receptor type C antibody
    • protein tyrosine phosphatase, receptor type, C antibody
    • Protein tyrosine phosphatase, receptor type, c polypeptide antibody
    • Ptprc antibody
    • PTPRC_HUMAN antibody
    • Receptor-type tyrosine-protein phosphatase C antibody
    • T200 antibody
    • T200 glycoprotein antibody
    • T200 leukocyte common antigen antibody
    see all

Images

  • Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells fixed in 4% PFA and stained with purified ab40763 at a dilution of 1 in 20 (red line).

    The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

  • Immunohistochemical staining of paraffin embedded human tonsil with purified ab40763 at a working dilution of 1/250.

    The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

    PBS was used instead of the primary antibody as the negative control (shown in the inset).

  • Immunofluorescence staining of Jurkat cells (Human T cell leukemia cell line from peripheral blood) with purified ab40763 at a working dilution of 1/100, counterstained with DAPI.

    The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel.

    The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

    The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40763 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

  • Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (purified) + HuT-78 cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

    Predicted band size: 147 kDa
    Observed band size: 200 kDa
    why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST

  • Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (purified) + Raji (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

    Predicted band size: 147 kDa
    Observed band size: 200 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST

  • Unpurified ab40763 showing positive staining in normal spleen tissue.

  • Unpurified ab40763 showing positive staining in normal colon lymphoid cells tissue.

  • Unpurified ab40763 showing negative staining in skeletal muscle tissue.

  • Unpurified ab40763 showing negative staining in normal kidney tissue.

  • Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (unpurified, blocked with 3% milk) + Jurkat (Human T cell lymphoblast-like cell line) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 147 kDa
    Observed band size: 200 kDa why is the actual band size different from the predicted?


    Exposure time: 8 minutes


    This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes.

    The membrane was then blocked for an hour using 3% milk before being incubated with ab40763 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Go here to learn more about KD:

    https://www.abcam.com/index.html?pageconfig=resource&rid=15749

     

References

This product has been referenced in:
  • Quispe Calla NE  et al. Exogenous oestrogen inhibits genital transmission of cell-associated HIV-1 in DMPA-treated humanized mice. J Int AIDS Soc 21:N/A (2018). Read more (PubMed: 29334191) »
  • Sherman H  et al. A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity. Front Immunol 9:857 (2018). Read more (PubMed: 29740450) »
See all 12 Publications for this product

Customer reviews and Q&As

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1-8 of 8 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (HL60 (human acute promyelocytic leukemia))
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Loading amount
15 µg
Specification
HL60 (human acute promyelocytic leukemia)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 30 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (MEL (murine erythroleukemia cell))
Permeabilization
Yes - Tween-20
Specification
MEL (murine erythroleukemia cell)
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Acetone

Abcam user community

Verified customer

Submitted Oct 30 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Human breast cancer tissue (form MDA-MB-231 Xenogr)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 6.0 Citric Acid
Permeabilization
Yes - Tween-20
Specification
Human breast cancer tissue (form MDA-MB-231 Xenogr
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 29 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (tonsil)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA ph9
Permeabilization
No
Specification
tonsil
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 20 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tonsil)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 9.0 EDTA
Permeabilization
No
Specification
Tonsil
Blocking step
PB ab64226 as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Mar 30 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (skin)
Permeabilization
No
Specification
skin
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 26 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (human small intestine tissue)
Specification
human small intestine tissue
Permeabilization
No
Fixative
Paraformaldehyde

Dr. Irit Shoval

Verified customer

Submitted Sep 19 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Sample
Human Tissue sections (Tonsil)
Specification
Tonsil
Permeabilization
No
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted May 31 2014

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