Hi, I have bacterial cell free supernatant which has an inhibitory effect to some pathogens and wanted to know if the nature of molecule in the supernatant is protein. I tried different proteases treatment but couldn't succeed. Could anybody suggest me other methods to characterising the different proteins?
Carry out an ammonium sulphate precipation and then do a SDS page electrophoresis
Dear Hanna,
I agree with the above answer. If you are sure that the supernatant contains proteins, make protein extraction followed by hydrolysis according to Lista et al. (2011). Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS. BMC Microbiology, 11: 267. The protein and peptide extract is then fingerprinted by MALDI-TOF-TOF MS, range from 0.2 to 150 KDa.
Alternatively, you can use LC Tandem Mass Spectrometry for identification of fractions.
Dear Hanna:
First of all culture supernatant will have many metabolic products that can inhibit other organisms, so broaden your look. I agree with Ashraf, LC-MS-MS ot MALDI-TOF-MS would allow you to look at all of the components. You would still need to purify without the loss of activity, so may be gel chromatography, TCA precipitation, using XAD2 for recovery of peptides with acetone would be a quick way to isolate peptides. All the best
Dear Dr. Nathan,
I had the privilege of meeting you in Ismailia, Egypt many years ago. Thank you for your valuable answer.
Dear all,
Thank you so much for the detailed and helpful explanation.
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