Hi,
I am using precast gel (Bolt 4-12% Bis-Tris precast gel) and in its specification the separation range of the gel is 3.5kDa - 150kDa.
I am running some protein samples with size of 300 - 400 kDa with that precast gel and the gel is not working well.
Does anyone know what should I do if my protein is not in the separation range of precast gel?
(The precast gel protocol says running the gel in 165V for 35 minutes but I tried 35 minutes to 1 hour (in 165V) and I could not find the large size of proteins in the gel matrix.)
This web page covers products for SDS-PAGE of very large proteins like yours.
http://tools.thermofisher.com/downloads/27965_HiMark.pdf
I am assuming your protein is a single polypeptide. If it is made up of several smaller polypeptides, you may be having other difficulties.
Hi Huihun,
the separation of high molecular weight proteins by SDS-PAGE is very diffcult, one
alternative is the usage of agarose gels for protein separation, below two links that
might be helpful
best regards
Bogdan
http://link.springer.com/protocol/10.1007%2F978-1-61779-821-4_10
http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_XIII_-_Protein_Separation_in_Agarose_Gels.pdf
Hi
A tris-acetat gel might also be an alternative for your study
https://www.thermofisher.com/de/de/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/nupage-protein-gels/nupage-bis-tris-gels.html
best regards
You probably can use SDS-Page gels with very low % of polyacrylamide and agarose (0.5%) described in some papers or eg a thesis related to titin (Neagoe, 2008).Agarose has to be around 50 deg and then mixed with the polyacrylamide solution.Just google and you might find other recipes in the muscle field, where large proteins like titin , nebulin, myosin have to be handled.The gels are easier to make than it sounds.
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