troubles with small proteins (less than 20) on SDS page - (Nov/30/2008 )

Hello guys!

need some help. i did running transfer and at the end there is no proteins in all lanes lower than 20KD.
marker did transferred to the memmbrane but after i didn"t see any small proteins in ECL i did ponce staining and there was no proteins lower than 20.

Could transfer did not work only on the proteins but not on the marker and could it go wrong only on lower part of membrane???

thanks.

check this link http://www.protocol-online.org/forums/inde...25&hl=small

may add 0.05% SDS to the transfer buffer

HI, There is two chance:
1- your protein has flow through the membrane. For how long you transfer:
2- protein doesnt attached very well to the membrane. What is the recipe of your transfer buffer?

I tried to run in 15% gel and this time the transfer worked (this put my protein higher in the membrain).

I ran it 100mV for 2h and the running was terrible. the loading buffer line was very thik and not straight and the bands in ECL appeared very thick (look like bricks).

What was wrong here. Why the bands so thick ?

try running our protein in a tris-tricine gel. this works better for low mw proteins and peptides. the bands are thick because tris-glycine gels work better for higher mw proteins.

you could also sharpen your bands by running a gradient gel but, for proteins <20kDa i recommend tris-tricine.

Thank u ! i wil try gradient gel first .