western blotting loading control - (Mar/03/2008 )
Hi,
I ran 50ug of protein from my whole cell lysates and probed for actin to use as a loading control. Unfortunately the transfer quality was poor at the bottom of my gel (it looks like I had a bubble). Is there a larger protein (around 100kaD) that I could probe for as a loading control?
Thanks!
jlolsen
Hi, i think actin is around 50 kDa. So, if you don't know what is that 100kDa protein, you can't use as control.
If you have some trouble, maybe is caused by large amount of protein loaded into. For example, in a gel of 1mm thickness, you have to not exceed 35-40ug of total proteins, otherwise you will have bad gel running and thus bad western.
So, could you tell me your conditions in your tranfer? So, i could help you better..
bye
S.75
If you have some trouble, maybe is caused by large amount of protein loaded into. For example, in a gel of 1mm thickness, you have to not exceed 35-40ug of total proteins, otherwise you will have bad gel running and thus bad western.
So, could you tell me your conditions in your tranfer? So, i could help you better..
bye
S.75
My gel is 7.5% acrylamide and 1.5mm thick. I run my gel at 70V until the samples pass through the stacking gel. Then I increase the voltage to 130V until the blue sample buffer runs off the gel. I transfer at 4*C and at 25V overnight onto a nitrocellulose membrane. I am not sure if my weird results are due to the running of the gel through the sample, or the transfer. I did notice that while my samples were running through the gel, they were not moving as a tight blue line. The blue sample buffer was wavy and spread out.
Thanks, if you had a wavy blue front, maybe is caused by excessive protein amount loaded in wells. I had this problem too and i resolved loading fewer protein amounts. But, 50ug could be right for a 1.5mm thick gel... so, could be running buffer as well, or separating buffer (controll its pH).
The tranfering conditions seem right.
Why do you use a 7.5% SDS-PAGE? do you need to look at a very high MW protein?
..there are some detergents in your samples?
bye!
S
I agree that 50ug is a lot to load per lane. I typically load around 10ug, but if the antibody is good you can load as little as 2 ug. If you need a heavier loading control, try Hsp90 (90 kD). We use the BD antibody and it's clean. However, since Hsp90 is a housekeeping protein, I've seen it's levels increase in response to cellular stress (ex. levels rise when I treat with MG132 proteasome inhibitor). It's still good if all your lanes are lysates under the same cellular stress, but it's not as good for comparing between stressed and unstressed conditions.