7 replies to this topic

[cupidstunt]

Hi!

I am trying to detect a small protein about 16kDa and am currently using biorad's a-bis acrylamide without much succes.. Somebody suggested me that Protogel apparently is better for small proteins.. I am not sure about this. Has anybody got any experience about this?

Thanks!

[bob1]

Use a higher percent gel or one with a low ratio of bis:acrylamide (e.g. 1:19)

[cupidstunt]

bob1, on May 12 2009, 02:17 AM, said:

Use a higher percent gel or one with a low ratio of bis:acrylamide (e.g. 1:19)

hi

I have tried using both 12% and 15% gels. I can get bands using a control peptide (positive control) but not my samples  

SO is it true that protogel is better than biorad's in this respect??

thanks

[bob1]

Are you talking about pre-cast gels? I pour my own, so I can't talk about the relative merits of different companies' pre-cast gels.

[cupidstunt]

bob1, on May 13 2009, 02:34 AM, said:

Are you talking about pre-cast gels? I pour my own, so I can't talk about the relative merits of different companies' pre-cast gels.

no I am not talking about pre-cast gels. I also make my own gels

[mdfenko]

have you tried the tris-tricine buffer system (shager and von jagow)?

it is better for separation of small proteins and peptides.

in fact, your 16kDa protein should separate nicely in a 10% gel with this buffer system.

you should be able to find it by searching the archives.

[cupidstunt]

mdfenko, on May 14 2009, 09:19 PM, said:

have you tried the tris-tricine buffer system (shager and von jagow)?

it is better for separation of small proteins and peptides.

in fact, your 16kDa protein should separate nicely in a 10% gel with this buffer system.

you should be able to find it by searching the archives.

Hi,

Thanks for the reply, yes I have tried using Tricine buffer, but with 12% gel instead. I haven't tried using 10% for that, wouldn't it be too large(pore size), for my small protein? Also, do you know if protogel is better for small proteins? I could not find any literature regarding the same

[mdfenko]

cupidstunt, on May 15 2009, 06:24 AM, said:

Hi,

Thanks for the reply, yes I have tried using Tricine buffer, but with 12% gel instead. I haven't tried using 10% for that, wouldn't it be too large(pore size), for my small protein? Also, do you know if protogel is better for small proteins? I could not find any literature regarding the same

it's not so much the pore size as the trailing ion that has the desired effect with tricine and acrylamide concentration of 10% or more. by the way, what was the result with tricine and 12% gel?

i'm not familiar with protogel so i can't properly respond to that part of your question.

Edited by mdfenko, 15 May 2009 - 08:18 AM.