When i try to check its expression on 18% SDS gel, i could not find any band. Could anybody suggest a method ?
If you have antibodies against this peptide, you can use simple use blotting. I dont know your expression system. But if you overexpress it in E. coli then you will purify it as well, what kind of tag you have? may be purification by using tag and then cleaving it by using protease (commercially available) can give you pure peptide. In this way at the end you have to purify the peptide by FPLC (gel filtration or ion exchange or HPLC RP). It is very necessary that this peptide during purification should behave properly and soluble, otherwise you will lost everything.
What about commerical synthesis of 21 amino acid long peptide. If you spend 400-500 USD, you will get 20-25 mg, >98% pure peptide. It could be a good option.
@Birendra singh: Thanks a lot for your useful suggestions. This small protein has not yet been well characterized. I wanted to check whether its expressed or not. Thanks again..
I do hardly believe that you will be able to express a protein of this small size in E.coli without any addition of a (cleavable) tag.
I will recommend you to synthesize this peptide and characterize its behaviour in different solutions to observe if can be folded properly. You can use circular dichroism for this purpose. Then if you have a functional assay to verify function of this synthsized peptide, do it. If synthesized peptide works, you dont need to express it. Or, expression in E. coli with tag and then cleaving with protease could be an option. However, if this peptide is soluble and correctly folded after separation from tag, then you will manage to get it.
I agree with Birendra. Probably best to simply make the peptide on solid-phase. It would take about a week to do this and it would be easy to purify by analytical or semi-prep HPLC. I think this would be much easier than trying the expression route. We routinely synthesize a 20 AA peptide on solid-phase in our lab with good yields and purity. Depending on how much you cleave, you should have plenty of peptide to do any characterization assays you need.
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