Validated using a knockout cell line

Anti-Integrin alpha 4/CD49D antibody [44H6] (ab220)

Reviews (1)Q&A (5)References (1)

Overview

  • Product name

    Anti-Integrin alpha 4/CD49D antibody [44H6]
    See all Integrin alpha 4/CD49D primary antibodies

  • Description

    Mouse monoclonal [44H6] to Integrin alpha 4/CD49D

  • Host species

    Mouse

  • Specificity

    Recognises the Integrin alpha 4/CD49D subunit, which associates with either Integrin beta 1 (to form VLA 4) or beta 7. The alpha 4 integrin is a 150 kDa molecule, which may be cleaved to 80 kDa and 70 kDa fragments.

  • Tested applications

    Suitable for: Flow Cyt, IHC-Fr, ICC/IF, IPmore details
    Unsuitable for: IHC-P

  • Species reactivity

    Reacts with: Human

  • Immunogen

    HOON pre-B leukaemia cell line.

  • Positive control

    • Flow Cyt: Human whole peripheral blood cells. IHC-Fr: Human tonsil tissue. ICC/IF: Wild-type HAP-1 cells.

  • General notes

     This product was previously labelled as Integrin alpha 4

     

Properties

Applications

Our Abpromise guarantee covers the use of ab220 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 1/40.

Use 10 µl of the working dilution to label 100 µl of human whole peripheral blood.

 

 

 

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

 
IHC-Fr 1/50 - 1/100.
ICC/IF Use a concentration of 5 µg/ml.
IP 1/20 - 1/25.
  • Application notes
    Is unsuitable for IHC-P.

    • Target

    • Function

      Integrins alpha-4/beta-1 (VLA-4) and alpha-4/beta-7 are receptors for fibronectin. They recognize one or more domains within the alternatively spliced CS-1 and CS-5 regions of fibronectin. They are also receptors for VCAM1. Integrin alpha-4/beta-1 recognizes the sequence Q-I-D-S in VCAM1. Integrin alpha-4/beta-7 is also a receptor for MADCAM1. It recognizes the sequence L-D-T in MADCAM1. On activated endothelial cells integrin VLA-4 triggers homotypic aggregation for most VLA-4-positive leukocyte cell lines. It may also participate in cytolytic T-cell interactions with target cells.

    • Sequence similarities

      Belongs to the integrin alpha chain family.
      Contains 7 FG-GAP repeats.

    • Domain

      The SG1 motif is involved in binding to chondroitin sulfate glycosaminoglycan and cell adhesion.

    • Post-translational
      modifications

      Phosphorylation on Ser-1027 inhibits PXN binding.

    • Cellular localization

      Membrane.
    • Information by UniProt

    • Database links

      • Alternative names

        • 269C wild type antibody
        • Antigen CD49D, alpha 4 subunit of VLA 4 receptor antibody
        • CD49 antigen like family member D antibody
        • CD49 antigen-like family member D antibody
        • CD49d antibody
        • IA4 antibody
        • Integrin alpha 4 antibody
        • Integrin alpha 4 subunit antibody
        • Integrin alpha IV antibody
        • Integrin alpha-4 antibody
        • Integrin alpha-IV antibody
        • Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA 4 receptor) antibody
        • ITA4_HUMAN antibody
        • ITGA4 antibody
        • MGC90518 antibody
        • OTTHUMP00000205320 antibody
        • Very late activation protein 4 receptor, alpha 4 subunit antibody
        • VLA 4 subunit alpha antibody
        • VLA-4 subunit alpha antibody
        • VLA4 antibody
        see all

      Images

      • Immunocytochemistry/ Immunofluorescence - Anti-Integrin alpha 4/CD49D antibody [44H6] (ab220)
        Immunocytochemistry/ Immunofluorescence - Anti-Integrin alpha 4/CD49D antibody [44H6] (ab220)

        ab220 staining Integrin alpha 4/CD49D in wild-type HAP1 cells (top panel) and Integrin alpha 4/CD49D knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab220 at 5μg/ml concentration and ab220272 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

      • Flow Cytometry - Anti-Integrin alpha 4/CD49D antibody [44H6] (ab220)
        Flow Cytometry - Anti-Integrin alpha 4/CD49D antibody [44H6] (ab220)
        Staining of human peripheral blood lymphocytes with ab220.

      References

      This product has been referenced in:

      See 1 Publication for this product

      Publishing research using ab220? Please let us know so that we can cite the reference in this datasheet.

      Customer reviews and Q&As

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      1-6 of 6 Abreviews or Q&A

      Western blot abreview for Anti-Integrin alpha 4/CD49D antibody [44H6]

       Good
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Human Cell lysate - other (blood Natural Killer cells)
      Gel Running Conditions
      Reduced Denaturing (12%)
      Loading amount
      10 µg
      Specification
      blood Natural Killer cells
      Blocking step
      Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
      Read More

      Abcam user community

      Verified customer

      Submitted Dec 17 2008

      Question

      Which portion of the integrin alpha4 that the antibody will detect? Is it the extracellular domain, or cytplasmic domain?

      Read More
      Answer

      Thank you for your enquiry. This product recognises the extracellular portion of integrin alpha 4. However, since epitope mapping was never done, there is no information on the exact epitope sequence.

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      Question

      I'd like to know if this antibody recognizes the N-term region of alpha 4 integrin or which part of the protein. Thanks in advance. Best regards

      Read More
      Answer

      Thank you for your enquiry regarding ab220. We have not mapped the epitope region recognized by this antibody therefore we do not know which region is recognized by the antibody. Sorry I couldn't help you more on this occasion,

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      Question

      follow up to previous enquiry: The two different positive controls were histag versions of human integrin alpha 4, both were loaded at approximately 50ng. It is possible there isn't enough protein in my samples but that doesn't explain why I'm not detecting my positive controls. Can you suggest a good starting dilution for this antibody also?

      Read More
      Answer

      Thank you for your email. We have had feedback from a customer who used this antibody at a 1:200 dilution for Western blotting. I'm really not sure why this antibody is not working for you, and can offer (if you have purchased the antibody in the past 90 days) a replacement vial to try or a refund or credit note. Please let me know how you would like to proceed.

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      Question

      follow up to previous enquiry: To answer your questions reqarding inquirey 735273 1. My transfer was good because my molecular weight markers transfer fine and I ran a number of gels at the same time and they all worked fine (different antibodys) 2. I found the lot number and it is 146463 3. I don't know the amount if any protein was loaded, I am determing mammalian cell transfection of the recombinant integrin alpha 4 from cell culture supernatant and am trying to verify expression.

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      Answer

      Thank you for your email. I noticed in your previous email that you mentioned for a positive control, "tried 2 different previously verified positive controls." Can you please elaborate on that? For your other samples, the soluble integrin alpha 4 secreted into cell supernatant, I wonder if there's enough present there to be detected by this antibody. Thanks again, and I look forward to hearing from you.

      Read More

      Question

      DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Soluble integrin alpha 4 secreted into cell supernatant, lacking c-terminus which is very small PRIMARY ANTIBODY AB220 mouse anti-human 60ul/15mls BSA==4ug/ml from a 1mg/ml stock Incubated overnight at 4C Wash in TBST 15min. 3 times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Yes, human integrin alpha 4/His ANTIBODY STORAGE CONDITIONS -20C SAMPLE PREPARATION 1X SDS running buffer heated 4 min. AMOUNT OF PROTEIN LOADED trying to determine ELECTROPHORESIS/GEL CONDITIONS Reducing gel 5-20% TRANSFER AND BLOCKING CONDITIONS Transfer 1 hour at 200V Block with 3% dry milk and no results Also tried BSA and no bands Have numerous good positive controls and none are showing up SECONDARY ANTIBODY goat anti-mouse HRP 1:2000 dilution in BSA 1 hr. Room Temp. Wash 3X for 15 min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Blocking reagents: tried 3% milk alone and BSA alone. Positive control: Tried 2 different previously verified positive controls

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      Answer

      Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. Did you determine how much protein was loaded onto the gel? Also, do you know if the protein transferred properly to the membrane? What was the batch number (located on the vial) or the Abcam order number or purchase order number that was used when you purchased ab220? Thank you, and I look forward to hearing from you.

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