I am essentially trying to separate native complexes using BNPAGE. I am trying to use precast tris-acetate gel 3-8% from Life Technologies.
I was wondering if it's a good idea to run the samples along with native running buffer (which is meant for BNPAGE, also from Life Technologies)? I think the buffer is a bis-tris containing buffer?
Hi Nikhil, in Native-PAGE it is important to know the isoelectric point of your proteins or proteins complexes to ensure that proteins migrate towards the electrode and to keep them from escaping of the gel. Based on this you have to choose the sample buffer enabling the correct charge of your proteins.
Dear Sandra, Many thanks for your reply. However, In my case since I am trying to separate entire cell extracts, Its rather not possible to choose precisely based on one complex. The reason I went to 3-8% Tris acetate gels is that I am interested in seperating the complexes around 700Kd and this percentage is ideal for that.
I have tried already using the conventional 3-12% Blue Native PAGE gels from Life systems and it works nicely for that, however the separation is not optimum.
Do you have any idea about the compatibility of Bis-Tris running buffer with a Tris-Acetate gel?
Dear Nikhil, I'm sorry but I have no experience with this system I always prepare my own polyacrylamide gels.
Hi Sven,
Many thanks for your reply. So you are indeed very correct. In fact with Blue native, the advantage is that you can include detergents like DDM/Triton and 6-Aminohexanoic acid in the buffer helps improve membrane proteins solubalization. Infact I tried one experiment with Tris-Acetate gel using HEK cell extract, and it just looks like crap. Now I understand that since the pH in the TrisAcetate gel is around 8-9, it will not work at all.
The issue is that we do not have a system to case gradient gels in the lab and it would take some optimization to have it running smoothly. The 3-8% gel would be ideal for my separation range, but alas there is no good manufacturer that sells it.
Do you think it helps to have a non gradient blue native gel? I read somewhere that this could even be better to seperate complexes that elute at the same height on a gradient.
sounds like something can be tried quickly. I will keep you posted.
Thanks a lot. Cheers
nikhil
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