I've blotted several samples under non-reducing conditions(because I have 2 non-reducing antibodies, CD63 & CD81) but these are both negative. Now I want to try staining with ALIX, but for that antibody my proteins need to be reduced. Can you incubate the blot with a DTT solution to reduce the proteins?
If you are running an SDS-PAGE and not a native PAGE gel then your proteins should already be reduced. The Laemmli loading dye has SDS and 2-ME so disulfide bonds are broken and the protein is denatured when boiled. You definitely don't want to include a reducing agent with your antibodies or they will fall apart and you will get no signal.
I've run an SDS-page gel, the point is the reducing agent. I need to reduce the proteins in the western blot, not the antibodies.
Instead of adding DTT to the sample before running the gel, is it possible the incubate the entire western blot iin a DTT solution to reduce the proteins?
it is but the point I was making is that the proteins should already be reduced by virtue of the SDS-PAGE sample prep. Its the same reason your other two antibodies (CD63 and CD81) didn't work.
you should be fine to go ahead with the staining as is. and leave out the 2-ME from the Laemmli dye and heat, not boil, when you prep your samples for the CD staining or do a native PAGE which is a total pain in the neck.
If you want to reduce proteins on the membrane use 2-ME just like blot stripping. I just don't know how well it will work because what blot stripping does is kill the HRP but the primary antibodies are still rock solid on the membrane.
your protein may end up reforming or never losing those disulfide bonds.
try it an let me know how it goes.
Thank you for your response Jason. The sample buffer we use( nupage sample buffer) does not standard include Mercaptoethanol, we normally add DTT. The CD63 and CD81 markers work only in non-reducing (no DTT or BME) samples, so no DTT was added. The antibody does work on the positive control and shows good signal, however not in the other samples. I will try to reduce the proteins using a DTT solution! So I can (hopefully) try ALIX normal antibody.
Hello Tim,
Please let me know if your experiment to reduced already transferred protein worked well. In our lab, we like to try something similar.
Venkatesh
Hi venkatesh,
I tried reducing proteins with a high concentration of DTT but it did not work for me !
When we reprobe a blot, we always use mercaptoeathanol at 50-55C for 30 min. This method is for removal of antibodies from the blot so that next antibody can be used for probing. However I needed to use dithiols to remove alkylation in our experiment. When I used both in combination and at 42C, it worked very well. I needed to remove alkylation to expose antigen on the blot. I hope this might help you. Good Luck.
- Badges
- Science method