Anti-Calpain 5 antibody - Aminoterminal end domain I (ab28280)

Dear Technical support,

Please help me with the following:
I have noticed that ab28280 from different lots behave differently. The one I previously received gave good bands; however I am struggling a lot with the antibody I most recently received. 1. I get a faint to none band, and sometimes I do need to incubate it for 2 nights at 4C even just to get a faint band. Do you still have the earlier lot), which I can have replaced with??
Thank you.

ANSWER:

Thank you for your reply.

As this is a rabbit polyclonal antibody, we would expect some lot to lot variation in optimal antibody concentrations. What are the conditions of your current WB protocol? I would like to see if there are any suggestions that can be made to improve your experience with this antibody.

Currently, we only have the lot you are already using, so a replacement from a different lot is not possible. If I am unable to make any protocol suggestions that improve your results with this antibody, I would be happy to offer another vial of this antibody from the current lot or a credit.

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.

I have few questions regarding antibody 28280 against N-terminal Capn5. Please help:
1. I understand that this antibody was produced using an immunogen Ab 41310, which to me appears that, is 1st 30 aaS of Capn5. But other than that, what other method was used to validate it as an antibody against Capn5. Was an immunoprecipitation conducted or may be the antibody detected band was sequenced to be Capn5? Anything else?
2. Do you have any positive or negative control to run with the samples of interest? May I be provided that?
3. The data sheet mentions, this antibody detects bands of 73 Kda and 68 Kda, and series of further cleaved bands. Full length Capn5 seems to be 76 Kda. I get a band along or slightly higher than 75 Kda. Please look at my blot attached to this email, and explain the band I am getting.
4. Also, could you please explain, what is the basis of getting 73 and 68 KDa bands, when it is against N-terminal?

ANSWER:

Thank you for contacting Abcam and I am very sorry about the delay in getting you this information.
Answers to your questions:
1 - We used lysates from A2058 cells, and from RD cells in Western blot to look for Calpain-5 protein. We don't see strong signals in most cell lines, as there is little calpain-5 there.
2 - Tissue lysates from colon, kidney, liver and trachea would make good positive control samples. I would recommend either ab29889 (human) or ab7265 (mouse) as good positive control samples if you wish to purchase one.
3 - I think the band you are seeing in your western blot is fine and it is running at the correct size (anything within about 5kDa of the predicted molecular weight is OK).
4 - You are correct that this antibody does not recognize amino-processed Calpain 5 and so you should not be seeing a band at 63kDa, and since you are not seeing this bad, I do not think it is a problem. I will have our data sheets team look at this and see if the reference to the 63kDa band should be removed from the website.
Once again please accept my apologies for the long delay in responding to you, this was due to us waiting on a response from the lab.
If there is anything else I can help you with, please let me know.