4 replies to this topic

[peanuts]

I wonder what is the range of protein amount that can be loaded for western blot?

I have no problem with position control (b-actin), but the band of interest is either nothing or a very faint band.

I tried to increase the concentration of antibodies, but without any luck.

I already load 65 ug protein, not surely if it is a good idea to load more (e.g., 100-150 ug)

Thanks for your help!

[casandra]

peanuts, on 25 July 2011 - 11:02 AM, said:

I wonder what is the range of protein amount that can be loaded for western blot?

I have no problem with position control (b-actin), but the band of interest is either nothing or a very faint band.

I tried to increase the concentration of antibodies, but without any luck.

I already load 65 ug protein, not surely if it is a good idea to load more (e.g., 100-150 ug)

Thanks for your help!

well, I think that there's no problem about loading a bit more as long as you don't overload the slot and spill to the neighboring lanes (you may have streaking though)...but perhaps what you may have to check out is the binding capacity of your membrane (200 ug? but am not sure) bec anything in excess will not bind anyhow. Couldn't you just enrich for your protein of interest? or try a different antibody or a fresh preparation.....also longer incubation time- 4 degrees O/N?

Edited by casandra, 25 July 2011 - 07:53 PM.

[Inmost sun]

the amount depends on the size of gel...for mini gels with 1 to 1,5 mm thickness, I think about 50 µg is the limit; it depends also on the proteins, pure protein to a single band or crude mixture of various proteins?

[peanuts]

Thanks for the reply. I did overnight incubation as well, but doesn't help.

The proteins that I loaded are crude proteins. I am not entirely sure about enriching my proteins. Is there any sound protocol that I can try out?

Edited by peanuts, 26 July 2011 - 06:05 AM.

[mdfenko]

we've loaded >100ug crude protein for westerns and had reasonable success.