Better resolution on SDS-PAGE then Western.... - (May/26/2006 )

simple question maybe....i run my protein on SDS-PAGE i get weak (but visible) signal on the gel of two bands....so i run western with very specific antibody (other controls are ok)....usually its 1000 times more specific then PAGE..... ...so I get bands on which i have to make my best of photoshop to see some faint faint bands which i lose on the print out... anyone has an idea what could have happened here???

Hi kathy,

I think I know what happened to your bands.
I think those proteins were not effeciently transfered from the gel onto the membrane. I was having the same problem myself not long time ago.

To be sure that is the case, I need to know some more things:
- what is the % of your gel?
- what is the size of the protein you are interested in?
- at which voltage and for how long have you done the transfer?
- What is the gel thickness?
- which type of membrane?

Is important that the gel % is adequate to size of your protein. A 12% gel is not the right % if you want to transfer efficiently high molecular weight proteins.

I good way to make sure your transfer is efficient is to stain your gel with coomassie blue after the transfer.
Is very easy procedure. Instead of throwing away your gel after transfer just incubate it with coomassie blue stain, while you proceed with blocking in your membrane.
This way you don´t loose time and know if your bands of interest stay or not in the gel.

You will solve the mistery.

hi kathy try increasin the pixel while you are saving the image. image should be saved in high resolution. also while you take printout it should be in high dpi so that your faint bands are visible.
it also depends on the efficiency of the scanner.
cheers