Hello,

cleaved caspase-3 is a small molecule, it is about 17/19 kDa, I recommend higer % of gel, check 13 or 15% gel. Your protein should be in the middle of the membrane.

good luck :)

As Ms. Andrulewicz-Botulińska already suggested, use a higher percentage of acrylamide, (i personally use 12% when checking for C-C3) to avoid that the bands unintendedly run out of your gel. Of course it depends on the antibody you use, i personally have good experience with the antibody #9664 from Cell Signaling. In addition, i always use the antibody #9665, which detects the cleaved and uncleaved C-3, as a control on a second membrane with the same lysates.

Sascha Seidel Edyta Andrulewicz-Botulińska

Thank you. :)

I'm using cell signaling 9661 as an antibody (1:1000 dil.) . I'm using the semi-dry system, and I think the problem may be during the transfer. I worked with 12V in semi-dry, but the proteins were not fully transferred. Do you have a suggestion for this system?

In fact, I would like to compare my own protocol with the protocol that you know works. I think I'm going to need little but important tricks. :)

Well, to be honest, no idea. Im usually using semi-dry blotting as well with an amerage of 100 mA per gel ( if you blot more than one at the sane time) for 90 min, but here results might vary depending on the system and transfer buffer you use. For semi-dry blotting i usually use buffer containing SDS, in contrast to wrt-blotting. I never had problems with the transfer, especially with such small proteins like the cleaved caspase. If at all, semi-dry blotting causes transfer problems with proteins over 100 kD in my case. Have you tried to do a Ponceau staining of the membrane to see if transfer is really the problem?

Sascha Seidel

Low weight protein ladder transfer was not sufficient. I haven't stained yet. But I'm going to do ponceau staining.

Thank you so much