Overview

  • Product name
    Anti-CD9 antibody [MEM-61]
    See all CD9 primary antibodies
  • Description
    Mouse monoclonal [MEM-61] to CD9
  • Host species
    Mouse
  • Specificity
    This antibody reacts with human CD9 antigen.
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cyt, Blocking, Functional Studiesmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Tissue/ cell preparation (Human). (Pre-B line Nalm-6).

  • Positive control
    • Prostate IHC-P: Human lung FFPE tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab2215 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 Use a concentration of 2 - 4 µg/ml. Use under non reducing condition.
IHC-P Use a concentration of 20 µg/ml.
Flow Cyt Use a concentration of 5 µg/ml.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Blocking Use at an assay dependent concentration. PubMed: 22745469
Functional Studies Use at an assay dependent concentration. The antibody induces FcgR-dependent platelet activation (aggregation).

Target

  • Function
    Involved in platelet activation and aggregation. Regulates paranodal junction formation. Involved in cell adhesion, cell motility and tumor metastasis. Required for sperm-egg fusion.
  • Tissue specificity
    Expressed by a variety of hematopoietic and epithelial cells.
  • Sequence similarities
    Belongs to the tetraspanin (TM4SF) family.
  • Post-translational
    modifications
    Protein exists in three forms with molecular masses between 22 and 27 kDa, and is known to carry covalently linked fatty acids.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Tetraspanin 29 antibody
    • 5H9 antibody
    • 5H9 antigen antibody
    • Antigen defined by monoclonal antibody 602 29 antibody
    • Antigen defined by monoclonal antibody 60229 antibody
    • BA-2/p24 antigen antibody
    • BA2 antibody
    • BTCC 1 antibody
    • BTCC1 antibody
    • CD9 antibody
    • CD9 antigen antibody
    • CD9 antigen p24 antibody
    • CD9 molecule antibody
    • CD9_HUMAN antibody
    • Cell growth inhibiting gene 2 protein antibody
    • Cell growth-inhibiting gene 2 protein antibody
    • DRAP 27 antibody
    • DRAP27 antibody
    • GIG2 antibody
    • Growth inhibiting gene 2 protein antibody
    • Leukocyte antigen MIC3 antibody
    • MIC3 antibody
    • Motility related protein antibody
    • Motility-related protein antibody
    • MRP 1 antibody
    • MRP-1 antibody
    • MRP1 antibody
    • p24 antibody
    • p24 antigen antibody
    • Tetraspanin-29 antibody
    • Tspan 29 antibody
    • Tspan-29 antibody
    • TSPAN29 antibody
    see all

Images

  • IHC image of ab2215 staining in human lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2215, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Overlay histogram showing Jurkat cells stained with ab2215 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2215, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Shi Q  et al. Differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells into endometrial cells. Stem Cell Res Ther 8:246 (2017). Read more (PubMed: 29096715) »
  • Gupta KH  et al. Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles. Dev Cell 41:674-684.e5 (2017). ICC/IF ; Human . Read more (PubMed: 28633020) »
See all 11 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Skin)
Permeabilization
No
Specification
Skin
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
Fixative
non

Abcam user community

Verified customer

Submitted Oct 30 2018

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 (Skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer, pH 6.0
Permeabilization
No
Specification
Skin
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 25 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Amnion Epithelial Cells)
Permeabilization
Yes - Triton-X
Specification
Amnion Epithelial Cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 22°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 15 2017

Answer

ab2215 should be used at a concentration of 2-4 ug/ml in blocking buffer when incubating your membrane whereas ab8216 should be used at a concentration of 2 ug/ml.

The gel should be run in non-reducing conditions as these antibodies do not recognise their antigens well when the western blot sample contains reducing agent. We therefore advise against adding beta-mercaptoethanol or DTT in the loading buffer.

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Answer

Thank you for contacting us.

The antibodies have not been tested for reactivity with bovine samples, and we have not received reports of bovine reactivity from our customers.

Please do not hesitate to contact us if you need any more advice or information.

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Question

BATCH NUMBER 163262 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM lots of bands and not at the right predicted molecular weight for cd9 SAMPLE as a positive control used full lysed DCs (NP40 lysis buffer, protease inhibitors: the samples however are the different fractions of a cell separation using a continuous iodixanol density gradient solution: trying to find a DC-SIGN/CD9 positive endosomal compartment. (used human DCs) PRIMARY ANTIBODY cd9 (Abcam) have tried it at different dilutions: 1/100, 1/500, 1/1000 dilutant: PBS/0.1% tween incubation time: 1 hour wash 3X for 10 min with PBS/tween DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED positive: full DCs ANTIBODY STORAGE CONDITIONS -20 C SAMPLE PREPARATION 15ul of sample (either fractions or full cell lysis) and 15ul of 2X loading buffer (no DTT) (and samples not heated) AMOUNT OF PROTEIN LOADED not exactly known, but in the full cell lysis there should be a lot of it ELECTROPHORESIS/GEL CONDITIONS non-reducing gel, 4-12% TRANSFER AND BLOCKING CONDITIONS transfer onto nitrocellulose, 100mA in transfer buffer blocking agent: milk powder SECONDARY ANTIBODY anti-mouse HRP (Sigma) in PBS/0.1% Tween (2ul/5ml) 1 hour wash 3X for 10 min with PBS/Tween HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? concentration of anitbody and of loaded protein (have loaded as much as possible), incubation times (have left it for less and longer than an hour) ADDITIONAL NOTES I have seen that you have another CD 9 antibody which is a rabbit anti human Ab, and I would like to perhaps try that one instead. I am doing a 3-month MSc project and thus have very limited time, which I cannot afford anymore to waste on trying to get an antibody to work. Thanks in advance.

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Answer

I'm very sorry to hear you are experiencing a problem with ab2215. I appreciate the urgency in resolving this problem due to the short time you gave in the laboratory, and I believe that with the following protocol tips you will have improved results with ab2215. Often non specific bands are due to the following problems: 1) protein degradation. we recommend the following inihibitors to be present in the lysis buffer: Aprotinin 2 µg/ml , Leupeptin 5-10 µg/ml, Antipain 2-10 µg/ml, Pepstatin A 1 µg/ml, Na-Fluoride 5-10 mM, phosphatase Orthovanadate 1 mM , PMSF 1 mM. If one of those has degraded in your samples this may explain the problem. Furthermore as you are performing a complicated fraction protocol this provides further opportunities for the proteases to act if they are not totally inhibited (please make sure all steps are carried out at 4C too). 2) the antibody is not binding to the membrane adequately. This can be due to too little or too much blocking (what percentage do you use and how long is your incubation for?). This can make a hughe difference in the binding ability of the antibody. I would recommend to try 5%BSA for 1 hour and incubating the membrane in TBST only overnight (TBST is better than PBST). The problem can further be due to too much primary antibody, so it is best to dilute the antibody more and incubate longer, to provide a slow but targeted binding to the protein of interrest (you may also find you can dilute the secondary more too). I hope the above recommendations will help you. Please do not hesitate to contact me again if I can be of further help,

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