Big size proteins in SDS-PAGE - (May/25/2006 )

Hi everyone!

I'd like to know if somebody has found the same problem when running big proteins (about 100KDa) in PAGE. I use a really specific antibody to develop western blot and I've detected that: when I use an 8%-acrilamide gel, this protein appears with his specific size, 100KDa; BUT when I use a 10%-acrilamide gel, my "before-specific-antibody" now detects a really intensiv band, but very much faster (lower size) than before!! I can't understand this. I've just studied a lot of conditions, like frozen samples, lesser boiling time, changing lysis buffer, ect.

Is it possible that in a more concentrated acrilamide gel happens some kind of electric phenomenons or higher temperatures that could destroy my protein and make it run faster?

Thank you very much.

Hi!
i'm not sure what is your problem.
in my opinion, i think higher percentage of gel can be seperate your protein subunit better than the lower.
And perhap if u use large gel and don't keep it on cool, high temp was an effect to your result.
so if u afraid of that will destroy your protein. try to run gel in refrigerator. In my lab , use that.

if you want to have the right size, you have to use the right percentage of acrylamide.
For each %, there is a range where the separation is linear and allows to calculate the relative weight.
for 100 kda, I would rather use 8 % than 10%

Hi!

Thank you very much for your help! I don't know what is happening, but I've tried to run my proteins in an 8% acrylamide gel again, and surprisingly I can`t find my protein. I only can see a very intensive and faster band than before. Maybe there's a problem with my Lysis Buffer or whatever. I'll continue searching information...

Bye!