control sample as a reference for Revers-phase HPLC - quantify and identify by RP-HPLC (May/14/2008 )
Dear Friends,
I want to use Reverse-Phase HPLC to separate my bacteriocin which it was extracted from free cell supernatant of overnight culture of my lactobacillus acidophilus.
I want to know what would be my control sample since I am working with unknown bacteriocin and I don't know what type bacteriocin or in what molecular weight range is inside of the extracted sample? is there any Standards available as control for this prupose in the Market for example for SDS-PAGE we have Protein standards with different range of molecular weight but how about if by RP-HPLC we want to find unknown bacteriocin? [I suspect that my bacteriocin is in low molecular weight (below 10 KD)]. what is control to run experiment to define it? do you have any idea about it since I'm new in this area. Thank you very much.
Leo
I want to use Reverse-Phase HPLC to separate my bacteriocin which it was extracted from free cell supernatant of overnight culture of my lactobacillus acidophilus.
I want to know what would be my control sample since I am working with unknown bacteriocin and I don't know what type bacteriocin or in what molecular weight range is inside of the extracted sample? is there any Standards available as control for this prupose in the Market for example for SDS-PAGE we have Protein standards with different range of molecular weight but how about if by RP-HPLC we want to find unknown bacteriocin? [I suspect that my bacteriocin is in low molecular weight (below 10 KD)]. what is control to run experiment to define it? do you have any idea about it since I'm new in this area. Thank you very much.
Leo
you dont have protein standards for RP-HPLC, but it is available for SEC-HPLC.You can separate your bacteriocin by ion exchange chromatography followed by RP-hplc. collect the separated fractions and subject the fractions in to MALDI-mass spec.
Thank you Prabhusankar. finally you replied my answer. I read some papers and found that as you mentioned the used cation exchange chromatography then applied to C18 column and followed by RP-HPLC. I will also check for MALDI-mass spec. as you said and see how the results. thanks again.
Leo