Overview

  • Product name
    Anti-Integrin alpha 2+beta 1 antibody [16B4]
    See all Integrin alpha 2+beta 1 primary antibodies
  • Description
    Mouse monoclonal [16B4] to Integrin alpha 2+beta 1
  • Host species
    Mouse
  • Specificity
    We have data to indicate that this antibody may not cross react with Rat. However, this has not been conclusively tested and expression levels may vary in certain cell lines/tissues.
  • Tested applications
    Suitable for: ICC/IF, IP, Flow Cyt, ELISA, IHC-Frmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Purified human beta 1 preparation from HT1080 fibrosarcoma cell extract.

  • Positive control
    • ICC-IF: A431 cells
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab30483 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
ICC/IF Use a concentration of 5 - 10 µg/ml.
IP Use a concentration of 25 - 50 µg/ml.
Flow Cyt Use 0.1µg for 106 cells.

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

 

ELISA Use a concentration of 10 µg/ml.
IHC-Fr Use a concentration of 30 - 40 µg/ml.

Target

  • Cellular localization
    Cell Membrane; single-pass type I membrane protein
  • Database links
  • Alternative names
    • CD29 antibody
    • CD49B antibody
    • Collagen receptor antibody
    • GPIa antibody
    • GPIIA antibody
    • Integrin alpha 2 antibody
    • Integrin beta 1 antibody
    • ITGA2 antibody
    • ITGB1 antibody
    • Platelet antigen Br antibody
    • Platelet glycoprotein GPIa antibody
    • Platelet glycoprotein Ia antibody
    • Platelet membrane glycoprotein Ia antibody
    • Very late activation protein 2 receptor alpha 2 subunit antibody
    • VLA 2 alpha chain antibody
    • VLA 2 antibody
    • VLA4 antibody
    • VLAA2 antibody
    see all

Images

  • Overlay histogram showing HT1080 cells stained with ab30483 (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 (ab30483, 0.1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse Alexa Fluor® 488 (IgG; H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HT1080 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • ab30483 stained in A431cells. The cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1 hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with ab30483 at 5 µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1 ug/ml overnight at +4°C. The secondary antibodies were ab150117 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

  • ab30483 staining human breast cancer cells by ICC/IF.  Cells were PFA fixed and blocked with 1% serum for 16 hours at 20°C prior to incubating with ab30483 (at 30µg/ml) for 16 hours at 4°C.  An Alexa Fluor® 488 conjugated goat anti-mouse antibody, diluted 1/200, was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Mrazkova B  et al. Induction, regulation and roles of neural adhesion molecule L1CAM in cellular senescence. Aging (Albany NY) 10:434-462 (2018). WB . Read more (PubMed: 29615539) »
  • Chung CH  et al. Aggretin Venom Polypeptide as a Novel Anti-angiogenesis Agent by Targeting Integrin alpha2beta1. Sci Rep 7:43612 (2017). Read more (PubMed: 28252668) »
See all 9 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Thank you for your reply.


Based on what you have already tested, I am not sure there are many more steps to be optimized. One consideration would be to extend the primary antibody incubation to overnight at 4oC. Longer incubation with less antibody tends to result in a cleaner, more specific signal. I am also happy to send out a new vial of antibody or issue a credit if you would prefer.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Colon-Ca)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric Acid 20mM
Permeabilization
No
Specification
Colon-Ca
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 02 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Prostate Cancer cells (PC3))
Gel Running Conditions
Reduced Denaturing (10% gel)
Loading amount
30 µg
Treatment
Anticancer drug
Specification
Prostate Cancer cells (PC3)
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 Jun 09 2016

Answer

Thank you once again for your telephone enquiry.

I am sorry to confirm that this antibody has been tested externally in ELISA and regrettably we have no further details to provide on the protocol on this occasion.

We aim to provide as much information as possible to customers, and I amsincerely sorry we have no more information to send.

If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for your enquiry.

I am sorry I am not able to confirm much further detail regarding the immunogen for ab30483 Anti-Integrin alpha 2+beta 1 antibodyas this particular product has been sourced externally.

However, I can confirm that in testing it is capable of immunoprecipitating a non-reducible alpha subunit from I125 surface labeled Human cell line extract. It is confirmed as specific to the alpha 2 subunit by relative expression of antigen on various cell lines by FACS, and its recognition of affinity purified alpha 2 beta 1 in dot blots.

I am currently waiting for further detailsregarding the ELISA testing and will contact you again with more information once I have it.

I hope this information will be helpful to you and thank youfor your patience. if you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for getting back to me with an update of your progress.

I have gone back and had a look at the previous results of your colleague. There definitely seems to be some staining of fibroblasts (in the boiled sample). As you say, it may be a case of optimising the antigen retrieval. It seems that the epitope recognised by this antibody is quite sensitive to fixation and the subsequent retrieval seems important. It may be worthwhile trying a few different conditions in order to obtain the optimim staining. As can be seen from the slide I have attached to this email, different targets and tissue preparationshave different optimum conditions which need to be evaluated.

The difference observed in your staining compared to the article I sent could be due to a number of different factors. It appears that the antibody used in this article is composed of three different monoclonal antibodies, recognising the integrin alpha2 chain andthe transmembrane glycoprotein which associates with integrin beta1:

http://www.funakoshi.co.jp/data/datasheet/EMF/M075-0.pdf

This in contrast to the antibody which you are using which is only able to detect one epitope of the integrin alpha 2+beta1. They have also used frozen sectionswhich is able to preserve thetarget proteinin a different way to how PFAfixationaffects thetissue.

One thing I would suggest may be worth trying now that the non-specific staining has improved is to remove the Tween (or any other detergent from the buffers used) as this will act to permiabilise the cells andpotentially this may bealtering thethe presentation of this membrane protein.

I hope these suggestions have been of help.If you have any further questions please do not hesitate to contact us again.

Read More

Answer

Thank you for providing that information. It has helped greatly in understanding what you have been observing.


To answer your question about what staining you can expect with anti-Integrin alpha 2+beta 1 antibody (ab30483) in human skin biopsies: unfortunately we have not performed a similar experiment ourselves and we have no journal articles or Abreviews where this antibody has been used for such purposes. I have however found quite a few references which have pointed to integrin alpha 2 beta 1 being expressed in the keratinocytes of the epidermis. The following reference has some very good images of the staining of the epidermis (which looks very similar to the staining you have been observing):

http://www.jbc.org/content/286/31/27804.full.pdf+html?with-ds=yes

And the following reference has also commented that the integrin isfound in the keratinocytes in the basal layeras well as other regions of the epidermis when wound repairand/or psoriasis is involved.


http://www.ncbi.nlm.nih.gov/pubmed/9536220


I therefore think the staining you have been seeing is what might be expected. There are however a few points in your protocol that I have noticed which may be adding to the non-specificity you have seen with the secondary antibody. Could you confirm, was the secondary which you used the same as what your colleague used to produce the staining you sent to me?


1. The length of time used for antigen retrieval seems quite long to me. We usually recommend trying 5, 10 and 15 minutes boilingto see which performs the best. I am however unsure of what the "reveal decloacker" is and it may be best to follow the instructions they have provided with it. Over doing the antigen retrieval can however lead to non-specificity and may be worth optimising.


2. You are using quite high concentrations of the antibody. Are you seeing good staining with 1/160 dilution? I would suggest trying the antibody at an evenhigher dilution, 1/250 and 1/500 and see if you still observe good staining. The dilution of the secondary antibody could probably also be increased, maybe try 1/500 and 1/1000 to reduce the non-specificity(but Invitrogen may be best placed to advise you on this).


3. I don't know what buffer you are using to perform these experiments but I would suggest using TBS (as it tends to give cleaner signals than PBS) with 0.1 %Tween added. This can reduce the surface tension allowing the reagents to cover the tissue evenly as well as reducing non-specific binding.


4. Is the secondary pre-adsorbed? If you are still having problems of non-specificity with the secondary after trying the optimisation tips it may be worth trying an antibody which has been pre-adsorbed. This process involves the secondary antibody being passed through a gel containing immobilized serum proteins from potentially cross-reactive species. The resulting purified antibody is more pure and specific, significantly reducing cross reactivity.An antibody such ashttps://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG1-heavy-chain-FITC-pre-adsorbed-ab98692.htmlmay be appropriate for the experiment you are performing.


I hope this information has been of helpand look forward to your reply.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Breast Cancer Cells)
Specification
Breast Cancer Cells
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C

Dr. Sharon Sneddon

Verified customer

Submitted Jul 10 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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