Anti-pro Caspase-12抗体(ab8118)

Thank you for your e-mail. Did you mean ab24852, rather than 24582? The antibodies are against caspase 12, not caspase 1. Can you please confirm that the customer is looking for caspase 12 and not caspase 1? I compared the two sheets of ab24852 and ab8118 and am asking my colleagues for an explanation of this important point. The two antibodies come from different suppliers and therefore we need to get in touch with them and find out why they can supply different products with the same image. It seems that it is the same product to me but I will confirm this. We have already noticed that ab8118 is recommended for human while ab24852 is not. Is the customer using human samples? Thank you for the customer's images. In the first attachment (1) there are two membranes. Is the top membrane the one probed with ab8118? Did it give other bands too? Is the bottom membrane the same? The two lots come from the same master stock 0302 so should give the same quality of bands. Is the customer using the same samples with the two lots or are they different? I look forward to hearing how the customer gets on with the recommended changes and more details on the blot and species of samples.

I'm sorry to hear your customer is experiencing problems with ab8118. The customer mentions that he/she provided images however these did not reach us, so I cannot see the membrane pattern with this antibody I'm afraid. It is very unclear what type of tissue was used and I would like to recommend a positive control of Human or Mouse spleen tissue lysate to make sure that the low signal at the correct band size is not due to low levels of the protein in the customer's samples. The protein is located at the ER and thus needs to be extracted with a strong detergent, a PBS buffer will therefore not be sufficient and I recommend to use a RIPA buffer for example, as the NP40 will lyse the cells very well. It is also very important to make sure that the right amount of protease inhibitors is added, and that those are fresh as they are often responsible for multiple bands unfortunately. The customer should also try a 1:500 dilution of the antibody, as recommended on the datasheet, to enhance his signal. I think an Ecl+ kit or more sensitive kits are more appropriate than Ecl, which is less performant. Regarding the problem of background bands, I would recommend to use 5% BSA for 1hr in TBST (not PBST) (Tween 0.1%) and to try to incubate the antibody in TBST only (and to try also with 1-2% BSA) (typically we incubate in TBST only but with some samples a little BSA in the antibody dilution buffer reduces the background). As the secondary antibody has not been tested alone I would suggest to try this, just to make sure that it is indeed performing well. I hope these recommendations will help. Please do not hesitate to contact me again should the customer still experience problems with those changes,

This antibody is raised against the N-terminal sequence AARRTHERDPIYKIKG. If this sequence forms part of the pro-peptide, then this antibody will be selective for the non-activated form. The blocking peptide for this antibody is available separately, ab8375.

These two antibodies ab8118 and ab8117 recognize the pre-activated form of caspase 12, however we have not tested for the activated/cleaved form.