By 'native' I mean no beta-mercaptoethanol and no SDS. Basically I would rather not disrupt any bonds that can be forming in terms of my protien of interest. I would rather make it in the lab than buy it, if possible. Thanks for any help!
The best advice that I can give is to obtain a good zymography protocol. You use non-reducing sample buffer (i.e. no mecaptoethanol) to dilute samples. SDS is however necessary in order to sufficiently charge proteins and allow for migration. In zymography the enzymes are "renatured" by washing with 2.5% (v/v) Triton X-100 solution.
I am not sure if this would reatach 100 percent of the broken bonds, but it allows for functionality of enzymes after SDS based electrophoresis.
If you want your protein complex to be preserved try blue native gel electrophoresis. You won't have to use SDS or beta mercaptoethanol.
Is it a native blotting you are talking about or just native electrophoresis? Do you want to test antibodies on native folded protein, or do you simply want to see complexes that the protein forms?
Blue natives gels are great for separating native complexes of proteins and there are native blotting protocols available. However BN PAGE also has its limitations, notably it is mostly used for membrane proteins. There are variations of it such as clear PAGE etc. I would recommend these articles for an overview:
Anal Biochem. 1991 "Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form." Schägger H, von Jagow G.
Anal Biochem. 1994 "Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis." Schägger H, Cramer WA, von Jagow G.
Nat Protoc. 2006 "Blue native PAGE." Wittig I, Braun HP, Schägger H.
Maria! I think blue native could also be used for proteins that are not membrane associated. Just the use of digitonin might vary. However feel free to correct me if I am wrong. Thank you.
Dear Paurav. Yes, you are right. Reason for BN PAGE being so popular for membrane-associated proteins is that coomassie binds to hydrophobic surfaces thus all membrane proteins migrate to the anode independent of the intrinsic pI.
However the dye binds also cationic amino acids on the surface of water-soluble proteins, which therefore can be resolved by BN.
Limitation is mainly for water-soluble proteins that do not bind coomassie and have a neutral/basic pI, so they can not be separated by BN-PAGE. Besides coomassie interferes with many fluorimetric and catalytic activity assays, as well as limit blotting efficiency. Still, some assays are not affected by the dye. Additionally cathode buffer can be exchanged during running for the one without coomassie, thus improving subsequent analysis.
There are alternative native PAGEs, namely clear native, that relies on the protein intrinsic charge, and high resolution clear native that uses detergent to impose charge on the protein.
Professor Steven Joseph Brookes, I do not get your answer, could you please explain the notion that whether the protein is denatured or not, the final blot will be the same? Thanks very much
After electrophoresis ,Transfer to blot using buffer with 0.04% SDS and with out methanol for 4h in ice.
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