5 replies to this topic

[Bindu]

Hello everyone

I have been working with proteins with molecular vweight of  150 Kda and 250 Kda.But I am unable to see the proper fragment  at the exact size as it gets stuck in the Stacking gel(4%).I am using Tris-Glycine gels of 8% resolving and 4%stacking portions and using overnight transfer at 25V with PVDF membrane at 4 degree temperature.Please let me know how to resolve this problem so that i can see a proper fragment of my protein.

[bob1]

Check the pH of the buffers that you are using for making the gels.  For 150 and 250 kDa I would recommend using 6% gels rather than 8% as the proteins will not resolve well at higher percentage.

[sinobiological]

we normally use higher voltage to transfer proteins. You can try 90V for 1-2 hours.
For our western-blot protocol as a reference, you can visit
http://www.assay-pro...ge=western-blot

[wanzybio]

Instead of going O/N incubation you can transfer for 1 hr at 40v,170mA.

[patrex2005]

Helloo all,
  I have problems with my samples (which are flushing fluids from mare´s uterus) , i use 2D-gel electrphoresis but the results were disappointed .Really,i dont know what should i do?
Anybody can help me please?
Thanks
Amgad

[bob1]

Hi Amgad - essentially you have said "I have a problem, what do I do" with no explanation of what your aim is or what you have tried (methodologically).