Anti-Securin antibody [DCS-280] (ab3305)

I have used this antibody in mouse, however the KO line is algo giving bands in WB.

ANSWER:

Thank you for visiting the Abcam booth at ASCB.

I am sorry to hear that you have experienced difficulties with our Securin antibody, ab3305.  I was hoping you would provide your protocol information for our records so that we may investigate this problem further.  Also, I would like to offer a replacement or credit for this defective antibody.  Please provide your previous order number along with your preference.

I look forward to your reply so that I may assist you further.  Please do not hesitate to contact us if you have any additional questions.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM We got a strong band ranging 65 kDa when we test this Ab against different mouse tissue extracts. We belive this band could be BSA. Was this antigen coupled to BSA?. The theoretical badn for securin looks to be a doublet but it?s very weak and there are many other bands.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ADDITIONAL NOTES We will run a control line with human cell extracts in order to see whether the background is the same we got in mouse extracts.

ANSWER:

The source of the antibody had further comments to your enquiry: We have not observed this 65kD band. Other references did not find this 65kD band either. Here are a few suggestions: It could be that there is high MW Pds1 present in your mouse tissue extract. Another likely possibility is that the F(ab) from mouse tissue extracts react with your secondary antibody and gives you a 65kD band.

I hope this information helps, please do not hesitate to contact us if you need further advise,

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM We got a strong band ranging 65 kDa when we test this Ab against different mouse tissue extracts. We belive this band could be BSA. Was this antigen coupled to BSA?. The theoretical badn for securin looks to be a doublet but it?s very weak and there are many other bands.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ADDITIONAL NOTES We will run a control line with human cell extracts in order to see whether the background is the same we got in mouse extracts.

ANSWER:

Thank you for your e-mail. I'm really sorry to hear you are having problems with ab3305. This antibody has not been tested in WB so it is difficult to know what the problem might be. Can you tell me more information about your protocol: dilution, buffers, tissues used, blocking agent, lysis buffer etc. This may help find out what the problem is.

The many bands may be due to too much primary or secondary antibody or to the type of blocking stage (test BSA or milk and choose which one give you a low background/less bands). It could also be due to a degradation of your samples if there isn't enough protease inhibitors. Try also to wash extensively in TBST between each step, and to quickly rinse the excess milk/BSA before incubating the membrane in primary antibody. A weak band may be due to a low amount of protein in your samples (securin is expressed at low levels in most tissues, except adult testis where is it highly expressed). The protein is nuclear and cytoplasmic, so choose a buffer which will extract securin at both levels. The theoretical band should be around 42kDa and the antibody should not recognise BSA.

Please do not hesitate to contact me if you need further advice, I hope the above suggestions will help you,