Anti-Perforin antibody (ab7203)

Hello

I have performed Western Blot with this antibody at the manufacturer’s recommended dilution. However, the antibody is not working properly. I am getting nonspecific bands all over the place. Is it possible to obtain a positive control from you? This way I may be able to titrate this antibody and get publishable results.

Thanks

ANSWER:

Thank you for contacting us and your patience, as we were closed over Easter.
I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.
As positive control, I can either recommend either fresh PBL or inflamed tissues, as well as the YT-Indy cell line - see either SwissProt or our datasheet of the ab86319 for reference: http://www.uniprot.org/uniprot/P10820 http://www.abcam.com/index.html?datasheet=86319
We normally do not send out positive controls for our antibodies. However we do guarantee all our products to work as stated on the datasheet. I would therefore suggest that we firstly investigate your results.
In order to do so, I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.
I look forward to receiving your reply.

We spoke over the telephone when I advised that I was using this antibody in WB and requested more information about a protocol. In particular, I am interested in how the cell lysate was prepared and whether the blot was performed under reducing or non-reducing conditions. Results so far show many background bands in addition to the expect band at 66kDa, but similar results with the -ve control lysate.

ANSWER:

We passed your enquiry on to the originator of this antibody and they have replied with the following response. If you have any further questions, please let us know. Many rat kidney models are known to contain anti-GBM antibody, which has approximately 20% cross-reactivity with anti-rabbit IgG. Since anti-rabbit IgG is the secondary Ab used to detect this rabbit polyclonal to perforin, there would be background if the sample was not well-denatured. A complete denaturing of the sample should improve the specificity.

I want to use anti-perforin(CAT ab7203) to block the cytotoxic activities of perforin by abdomal injection. Did you perform similar experiment with this reagent? And how much dose could you please give some advice?

I am looking forward to hearing from you soon. Thank you very much.

ANSWER:

Thank you for your enquiry and yiur interest in our products.

Abcam must draw your attention to the disclaimer as set out in its Terms and Conditions: " FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE", and it cannot endorse or otherwise encourage you to use its products for uses other that research.

This particular antibody has only been tested for Western blot application.

I know that all of the previous questions have been related to flow cytometry, but the most recent is one year old. Has it been tested for flow cytomtetry in the last year?

ANSWER:

Not to the best of our knowledge.

Need to use antibody to assess expression of Perforin in Human cells by flowcytometry/ by ELISA in culture supernates.

ANSWER:

The anti-rat perforin has not been tested for use in flow cytometry. Cross reactivity with other species has also not been determened.