Assume you have a protein heterodimer bound by a single disulphide linkage( monomers sizes 100kDa and 75kDa). How many bands will be observed in a) native PAGE b) Denaturing PAGE? What will be the relative position of bands in each technique?
P.S.: Denaturant used is Urea.
a/ One band (provided the heterodimer assumes one conformation that migrates as one band). If you add a reducing agent, it will migrate as two bands (or more if more than one conformation for each monomer is possible).
b/ What is "native denaturing" PAGE? Typically, it is either "native" of "denaturing" but not "native denaturing". If it is a denaturing PAGE with UREA without reducing agent(s), you would see one band (to see two bands you need to add a reducing agent, e. g. DTT, b-mercaptoEtOH).
Hi Mahesh,
I strongly agree with Libor because the native form of your protein will only run as a monomer under non-denaturing condition and run more than one(dimer or trimer) in the presence of a reducing agent like SDS .However, if you use 2-BETA MERCAPTOETHANOL as a reducing agent , it will help to break the disulphide linkage that bound the heterodimer of the protein.In addition using urea(depending on the concentration used) can denature your protein from heterodimer to monomer which might give you a band or two(also depending on the % and type of gel you use.
I hope this will help you.
All the best!
1. As pointed, there is not a thing called "native denaturing"
2. SDS is not a reducing agent but a denaturant in electrophoresis
3. Urea can not "denature" your protein from heterodimer to monomer if disulfide bonds dictate the generation of the heterodimer. Urea is a denaturant in electrophoresis that will denature the heterodimer as a whole (again if the struture depends on disulfide bonds)
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