I am doing Native page for purified mouse proteins (40kDa and 11 kDa), His-tagged at N-terminal....pI of my proteins are 6.1(40 kDa) and 5.33 (11kDa) . I performed 16% native gel (with stacking pH=6.8 and resolving pH= 8.8; without SDS ) but only observed 11 kDa protein. Higher mol.wt.protein was not entering the gel. After that I was told to run a 7% acidic Native gel (beta-alanine/acetic acid) but still the problem is same. 40kDa appears on SDS gel but stays in stacking of native gel (checked by western blot). Any suggestion would be highly appreciated.
Regards
Ashutosh
Ashutosh Vashisht ,
This might be a long shot but it has been shown that Ni++ promotes the oligomerization of some His tag-bearing proteins (see e.g. PMID 9668967 and PMID 16904956). Can you try adding some EDTA to the sample and checking if the problem goes away?
Also, if you have the required hardware and columns, it might be easier/faster to characterize the proteins by gel filtration. Native PAGE can be tricky.
Hope it helps!
I agree with Alejandro Martin that you should try gel filtration chromatography, because it will tell you unequivocally whether your protein is aggregated, which could be the reason it does not run into the native gel.
Dear Alejandro Martin,
Thank you so much for the response, but i didn't get the Ni++ thing here. I have done fplc purification of my proteins and there was no aggregation at that time.Before that i purified the proteins from inclusion bodies using 100 mM Tris, 5mM EDTA and 1 mM PMSF and dissolved the pellet in 100mM Tris-2M Urea. After that I exchanged the buffer to 20mM Tris,1mM EDTA,1mM reduced glutathione, 0.1mM oxidised glutathione,10 % sucrose. Actually I want to check the PPI through native.
Hi Ashutosh Vashisht - I had a similar problem with one of my bacterial proteins (45kDa). We used the same test with EDTA as suggested and that solved the problem to some extent but there was still residual protein in the stacking gel. We solved the issue by changing out the metal for IMAC which in our case appeared to prevent the aggregation. Since we were using the protein for crystallography we did size exclusion after which as per Alejandro Martin will give you an answer regarding your aggregation after your IMAC.
Dear Blake Balcomb,
Thank you so much for the inputs. I will surely do it with the EDTA. How much final concentration of EDTA should I use for my 5X Native sample buffer (100% Glycerol; Tris 0.5M-6.8 pH, Bromophenol blue 0.1%).
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