I tried to detect HIS tagged gp120 using capture ELISA. There was no color development. Could it be that the protein I purified was not gp120? The gp120 was NiNTA purified and has a PADRE sequence. I also used SEC to purify it further. I measured the concentration using nanovue spectrophotometer and both the SEC profile and nanovue indicate there was a protein. Should I try western blot as an alternative?
what's your purpose of the expression the gp 120? if you only want to make sure that the gene or the ORF of your gene , The WB is need, if there are some other purpose like to use in POCT and RAD,the ELISA or coillied gold may need to test with the commercail product..
good luck!
Thank you. I need gp120 for some immunization experiments. I think I will do a western blot. But could you tell me if not seeing colour development in ELISA could have been because the protein was not expressed properly by my cell line?
And could you suggest what alternative methods there are to detect presence of the gp120 other than western blot and ELISA?
For your ELISA, do you have positive controls to ensure the assay itself is working? If it still does not work, maybe at least one antibody does not recognize the native (or maybe not correctly folded) form of the protein. That's why I'd suggest a Western.
Do you see something of the expected size in a coomassie or silver stain? Ultimate confirmation (or disappointment :( ) may be done by a tryptic digest, followed by LC-MS). Another option is a qPCR of the mRNA.
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