I am optimizing a new antibody and it looks like my antibody is binding to the molecular weight markers, because I can clearly see them together with other bands on the final picture.
I dont have a big experience with western blotting but it is the first time I get this result and I have no idea about what is happening.
I use a horseradish peroxidase conjugated secondary antibody and chemiluminescence substrate, exposure time of 30 seconds. Ive tried primary antibody solution 1:1000 and 1:500 secondary antibody solution 1:2500 and 1:5000, 2% milk as blocking buffer, wash in TBST.
I dont get a very good signal for the band I am looking at but theres a very strong signal for the markers, which I know that are not supposed to be seen on this final picture (unless I want to get the negative picture).
I hope that this has already happened to someone, and I would be very greatful if someone could help me find a solution!

Thanks a lot

Bruno

It is not unusual to get non-specific (sometimes quite strong) cross-reactivity with the Molecular weight Standards. You can try to goof around with antibody concentrations and buffer/wash conditions, or you might try a different brand of MW marker. Sometimes switching the host animal for the secondary antibody can help.

If you think the strong signal from the markers are over-exposing your blot before you can get a good visualization of your protein of interest, you can try loading the marker in an end-well and then cutting it off the blot prior to incubation with your anitobodies - of course, use a ball point pen or something to mark the location of your standards. This is really easy to do if you are using pre-stained markers.

If the background is coming from the primary antibody, removing the markers prior to incubation may also increase the signal of your protein of interest.

thanks a lot, I think I will try to cut the markers off before incubation with antibodies.
I hope it will work better because the deadline for handing in my project is quite close!

Thanks

Bruno