Troubleshooting Protein Purification using FPLC - (Apr/07/2011 )
Hello,
Ive been working on purifying proteins for a while now, but I encountered a small problem. It seems like when i have my sample and I load it onto the fplc, it never sticks to the column and ends up in the flow through or end peak via 100% Buffer in the B Line. Since I'm running a labeled sample... is it still salvageable whatever contents are in beginning injection peak and final peak? I really dont want to lose the sample since it's isotopically labeled. any suggestions?
-ninjakid-
ninjakid on Thu Apr 7 22:53:08 2011 said:
Hello,
Ive been working on purifying proteins for a while now, but I encountered a small problem. It seems like when i have my sample and I load it onto the fplc, it never sticks to the column and ends up in the flow through or end peak via 100% Buffer in the B Line. Since I'm running a labeled sample... is it still salvageable whatever contents are in beginning injection peak and final peak? I really dont want to lose the sample since it's isotopically labeled. any suggestions?
Hola, yes you could pool the 100% peak and by other hand the flowthrought, and concentrate for ultrafiltation or dilute to low the concentration of something that avoid the bound to the colum. Is a nickel colum ?, if yes the 100% peak are multimeric forms or aggregates, if the protein of flowtrhough doesn´t bind could be because you are working at an acid pH, or the imidazol concentration in the sample to avoid inespecific interactions is quite high. Buena suerte, more people here will help you
-protolder-