Overview

  • Product name
    Anti-CD45 antibody [MEM-28]
    See all CD45 primary antibodies
  • Description
    Mouse monoclonal [MEM-28] to CD45
  • Host species
    Mouse
  • Specificity
    Human CD45 antigen (LCA). This antibody reacts with all alternative forms of CD45.
  • Tested applications
    Suitable for: Flow Cyt, IP, IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Tissue, cells or virus corresponding to Human CD45. Human thymocytes and T lymphocytes.

  • Positive control
    • Jurkat human leukemia T-cell lysate; Kg-1a human leukemia cell lysate, Human peripheral blood mononuclear cells

Applications

Our Abpromise guarantee covers the use of ab8216 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
Flow Cyt Use 1µg for 106 cells.

(Recognizes an extracellular epitope).
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IP Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Antigen retrieval is not essential but may optimise staining.
WB Use a concentration of 1 µg/ml.

Prepare and run samples in non-reducing conditions.

ICC/IF Use a concentration of 10 µg/ml.

PubMed: 23908685

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

  • Immunocytochemistry/ Immunofluorescence analysis of CTC isolated from mRCC patients stained for hematopoietic cells labeling CD45 with ab8216 (green). The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween. Subsequently, cells were stained with DAPI for 10 min, mounted with anti-fading medium and stored in the dark until evaluation. Left: CD45, right: DAPI, bottom: merge.

  • Ab8216 staining human normal tonsil tissue. Staining is localised to cellular membranes.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Immunocytochemistry/ Immunofluorescence analysis of citrated peripheral blood hematologic cells taken from head and neck squamous cell carcinoma (HNSCC) patients labeling CD45 with ab8216 (green). The staining method included fixation of the cells in 4.5% paraformaldehyde for 15 min, washing in PBS, permeabilization with 1× Perm/Wash Buffer for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 µg/ml) overnight at 4°C, wash in 0,1% Tween, binding of Cy3-conjugated secondary antibody for 30 min at 37°C, washing in 0,1% Tween.

  • Immunocytochemistry/ Immunofluorescence analysis of hematopoietic cells labeling CD45 with ab8216. The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween.

  • Overlay histogram showing peripheral blood lymphocytes stained with ab8216 (red line). The cells were incubated with the antibody (ab8216, 1µg/1x106 cells) for 30 min at 4ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/200 dilution for 30 min at 4ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed gating on peripheral blood lymphocytes.

  • Immunocytochemistry/Immunofluorescence analysis of human peripheral blood mononuclear cells labelling CD45 (green) with ab8216 at 10 μg/mL. Nuclei were counterstained with DAPI (blue).

References

This product has been referenced in:
  • Huo Z  et al. The relationship between allergic status and adenotonsillar regrowth: a retrospective research on children after adenotonsillectomy. Sci Rep 7:46615 (2017). Read more (PubMed: 28418014) »
  • Jackson MV & Krasnodembskaya AD Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC). Bio Protoc 7:N/A (2017). Read more (PubMed: 28534038) »
See all 22 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Question
Answer

Thank you for contacting us.
There are 3-8 molecules of biotin at each antibody molecule.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answer


IF by definition refers to any antibody application that utilizes fluorescence as the detection method. Thus, technically ICC and IF are one in the same, and you could argue that IHC is also a form of IF.

For the purposes of searching for products on our website, we distinguish those antibodies that have been tested in IHC with IHC followed by a letter designation (P-paraffin embedded sections, Fr-frozen sections, FoFr-formaldehyde fixed frozen sections). If an antibody has ICC or ICC/IF listed under tested applications they are the same thing and guaranteed to work in immunocytochemistry.

It sounds like you are performing IHC-P, immunohistochemistry on paraffin embedded sections; thus, all three antibodies that you identified, ab53003, ab8216, and ab111250 are tested and guaranteed to detect their respective targets via IHC-P in human samples. As stated in our AbPromise guarantee, we are happy to provide scientific support, replacement or refund should these products if they do not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Pancreatic Cancer)
Specification
Pancreatic Cancer
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: CC1, a Tris/Borate/EDTA buffer pH 8
Permeabilization
No
Blocking step
Inhibitor CM as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Dec 18 2012

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM no band-->even at different concentrations (range from 1:500-1:1000) SAMPLE human Hodgkin-&primary mediastinal B-cell lymphoma cell lines ( L428, L540, HDLM-2, KM-H2, L1236,MedB-1) human tonsil whole protein extract in RIPA lysis buffer PRIMARY ANTIBODY ab8216, anti CD45, mouse monoclonal tried almost every dilution between 1:500 and 1:1000 in milk or only TBST over night incubation at 4?C DETECTION METHOD pierce: super signal west dura POSITIVE AND NEGATIVE CONTROLS USED positive control: human tonsil ANTIBODY STORAGE CONDITIONS 4?C short term storage SAMPLE PREPARATION in RIPA lysis buffer NuPAGE LDS sample buffer heated for 10 Min at 95?C AMOUNT OF PROTEIN LOADED 100?g ELECTROPHORESIS/GEL CONDITIONS 4-12%Bis-Tris gel MOPS running buffer TRANSFER AND BLOCKING CONDITIONS transfer buffer (invitrogen) blocked in 5% milk SECONDARY ANTIBODY pierce - secondary anti mouse antibody (HRP conjugated) dilution: 1:1000 incubation: tried 1h at 37?C to 2,5 hours at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? dilution time of incubation, temperature while incubation buffer blocking ADDITIONAL NOTES I'm a little upset this antibody does not work. CD45 is a real routine antibody for IHC and now it does not work for western blot? Please help me with my problems. I have had enough problems with abcam antibodies such as SHP-1

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with ab8216. I have read your technical questionnaire and I have a few comments. You are using an extraction protocol that I would advise: RIPA buffer. I also consider your choice of a positive control good in the form of human tonsil. I would not recommend using as much as 100ug in western blotting. However, given that you are obtaining no signal whatsoever, please can you tell me whether you have confirmed the successful transfer of your blot by Ponceau S. Also can you tell me whether you have confirmed the integrity of your lysate preparation using an alternative CD antiserum using these conditions. I look forward to hearing from you.

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Answer

I am sorry to hear that you have been experiencing problems with ab8216 in Western blotting. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 90 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered. Below I enclose a link to a questionnaire which will help you put your protocol information together very easily so we can look into the details of the experiment and provide some help. Thank you, and we look foward to hearing from you. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=8216&mode=questionaire

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Answer

We do not know the epitope or if this antibody recognizes any fragments less than the expected size of the protein. We do not have access to the original WB data. We did not perform such a study, and most likely, if we did and saw any smaller fragments, we would consider it as non-specific proteolytic cleavage during sample preparation rather then as a result of some biological process in a cell. Moreover, the major bands migrate between 180-220 kDa, so any cleavage fragment would presumably be lost due to the low density of gel used for LCA isoforms (so the fragments would migrate with the front of the gel). In short, you should test this hypothesis in your particular experimental setup. If you have any additional questions, please contact us again.

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Answer

ab8216 has not been tested in mouse, so we have no further information on this. We have several CD45 antibodies so I suggest that you conduct a search through the Abcam Website. I also recommend that you should click on the links “Product Reviews” and “Technical Enquiries” both accessible via the green menu bar on the datasheet. Here you will find customer reviews and past questions and answers specifically relating to that product. If you cannot find what you are looking for within the Abcam catalog, I suggest that you click on the link to "The World's Antibody Gateway". The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antibody companies (currently 249).

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Answer

This antibody has not been tested for cross reactivity with sheep. As noted on the datasheet, for immunohistochemistry, no treatment of parrafin sections neccessary. I suggest that you examine the degree of sequence homology between human and sheep CD45. We do not have an antibody agaisnt CD45 that has already been tested for cross reactivity with sheep, but I suggest that you conduct a search through the Abcam home page, and click on the link in the yellow menu bar to "The World's Antibody Gateway" to determine whether any other companies stock such an antibody. The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antibody companies (currently 169).

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Question
Answer

No. Even if this antibody did recognise mouse CD45, the antibody has been raised in mouse, therefore the anti mouse secondary would non-specifically bind to the tissue leading to high background staining. At current we do not stock an antibody towards CD45 that has been raised in any other species.

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