Protein size and gel percentage - (Nov/26/2009 )

Hi all!
Could anyone kindly help me finding a reference for guidance of which gel percentage to use in connection to sizes of proteins to be resolved on SDS-PAGE and why? In other words, I have to prove my boss by the respected reference that it is not OK to SDS-PAGE on 4-12% to see a protein (after western and antibody staining) with calculated moleclular mass of 14 kDa (though usually runs near 16 kDa). I try to persuade her that 15% is better and protein should be in the middle of gel and not at the end, while she says that if I stop PAGE in a good time (before dye goes out of gel), then it is acceptable. The basic problem that for some resasons I don't know, the Westerns from 4-12% and 15% look different, whereas she likes more the 4-12% since it fits with her theory while 15%-doesn't.
Thank you very much for help!
P.S. Don't think that I didn't try to find it myself. Thanks again!

the westerns will look different as there is a gradient of pore size. the molecule will travel moer in the 4-12% than in the 15% as the pore size is larger at the start of the resolving gel!!!
but ya for a protein of 14 kDa 4-12 is not a good option.. you can in fact try a 8-20 gradient or choose if 15% is better.. the basic point is to be able to see the HMwt impurities and LMwt Impuriteis with good resolution!!!
I wonder why your PI is so stubborn abput this simple scientific fact!!!
Good luck to u though!!!

Hello!
Do you have any references? She is immunologist (no clue about biochemistry) and the type that trust only some references and not just my words. Thanks!
It is not just westerns look different: the band of interest, which she does not see on 4-12%, shows up on 15% (it's about treated and untreated samples). The latter is what she doesn't like, since it is against her theory.

Pradeep Iyer on Nov 26 2009, 12:00 PM said:

Ya i understand.. u can show her the sambrook molecular cloning volume 3.. it gives the sizes and percentages of gels to be used. but i fear the gradient ideal percentage wont be given there and it is a rare find.. still if i come acroos one will post it... but the homogenous percent can be fount in sambrook and many online sites too!!!

http://www.fermentas.com/en/support/application-protocols

its clearly given here that a 4-12 cannot be used for 14 kDa molecule. This shud be enough to shut your PI i guess. Its on the fermentas website!!
Application protocols - protein electrophoresis and analysis - SDS-PAGE

Protein MW range, kDa Recommended gel, %
~5-50 18 %
~5-60 16 %
~10-80 14 %
~20-150 12 %
~30-200 10 %
~40-250 8 %
~60-300 6 %
~100-400 4 %
Protein MW range, kDa Recommended gradient gel, %
~5-100 10-20 %
~5-300 4-20 %
~10-200 8-16%
~30-300 4-12 %

Thanks so much for that info! I've been trying to find it!!!
So, I have been doing western blot for an 8.7KDa protein in a 4-12% Bis-Tris gel and I have been able to detect it.

Pradeep Iyer on Nov 26 2009, 05:50 AM said:

THANK YOU!!!!!!!!!!! This is EXACTLY what I needed!!!! Have a nice... everything!

Pradeep Iyer on Nov 26 2009, 12:50 PM said:

thanx ktk... u tooo have the nicest of everything and may it be more easier to convince ur PI now on!!!