Anti-Integrin alpha 2+beta 1 抗体 [16B4] (ab30483)

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.

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.

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.

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.

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.