GST-Tagged protein - (Mar/26/2009 )
hiiiiii,
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
-sssss-
sssss on Mar 26 2009, 08:45 PM said:
hiiiiii,
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
your protein could be degraded.
-Nrelo-
try adding a protein cocktail, PMSF, aprotonin and any range of protease inhibitors in your lysis buffer.
-MaggieRoara-
Yeah...even I think its degradation of protein....what temperature did you inubate it at for 16 hours?
This may be killing it.
Best,
TC
sssss on Mar 27 2009, 10:15 AM said:
hiiiiii,
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
-T C-
T C on Mar 27 2009, 03:37 AM said:
Yeah...even I think its degradation of protein....what temperature did you inubate it at for 16 hours?
This may be killing it.
Best,
TC
This may be killing it.
Best,
TC
sssss on Mar 27 2009, 10:15 AM said:
hiiiiii,
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
I hav a problem in cutting GST-Tag from my protein.After incubating GST-tagged beads with precision protease for 16 hours, theoretically I should get a clean single band. But in my coomassie gel there are multiple bands.In western I am geting one more extra band below the size of my protein.How will I get a single band????
thanx 4 reply...I incubate the GST-Tagged beads with precission protease at 4 degree
-sssss-