I need to form and disrupt the beta sheets just by adding some external agent.please give me detailed work references and some help from your experiences as well.
An 1 % sodium dodecyl sulfate (SDS) buffer, an anionic detergent, will denature most proteins.
especially with you reduce with beta-mercaptoethanol or DTE or DTT too.
if you in addition boil the mixture for some minutes you can be fairely sure that the structure is disrupted.
Guanidinium thiocyanate (GITC) or urea will also di the trick, but here you have to use molar concentrations.
In addition to urea or sds protocol suggested by Pal you may also try HFIP disaggregation and then redissolve in buffer containing high amount of denaturants
Beta sheet proteins are particularly resistant to SDS denaturation (see for example http://pubs.acs.org/doi/abs/10.1021/bi0491898). Besides, SDS unfolded states contain significant amounts of residual structure.
If you want near-complete unfolding, I suggest using urea or guanidine chloride as denaturants (at molar concentrations as indicated by Pal).
thank you friends and professors i have been following your views practically and they helped me but after few minutes my compounds are degrading in to byproducts .how to avoid this problem?
Hi Kishore,
Partially unfolded states of proteins are good substrates for proteases and other chemical cleavage agents. If this were the case, you can try to avoid its presence (e.g. by improving the purification protocol), or to reduce the reaction rate (e.g. by lowering temperature, increasing viscosity or using specific inhibitors).
By the way, the contrary can also be true. Partially unfolded states of proteins are often prone to aggregation.
Hi Kishore
In addition to Dr González, you should add some enzyme inhibitor, like Complete or PMSF.
pH, in combination with GdnHCl and GndSCN, plays an important rule in aggregation state of partially unfolded proteins , make sure that the pH of your preps are good for your experiment.
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