Is that from single band of protein by SDS-PAGE where we confirm or any other method?
Thank you in advance.
I agree with Masood, SDS is an easy method giving lot of surety regarding the purification of your enzyme. Well, Iso-electric focusing can further tell regarding the purity of the enzyme. In addition, Chromatography (Size Exclusion, ion exchange or reverse phase) and mass spectrometry can also be used.
Hope this will help
For the preliminary examining, you can use Bio-Rad protein assay for quick test. If you use beads for purification, you can also check beads after enzyme purification to make sure there is nothing on the purified beads. This method is cheaper and take only 10 min for examination.
If you getting single clean band in SDS PAGE then better option is Go for MASS Spectrocopy (ESI MS or MALDI MS). Or HPLC is another good option
SDS-PAGE is the best method to find your sample purity.
After each purification method, you must run a SDS-PAGE. When you find one band on your gel, you can approximately say "my protein is pure".
"Approximately" means that in very limited reports, there were some proteins which had similar Mw and concentrated in one band on SDS-PAGE. For these proteins, you can run IEF to separate them.
It depends on which further experiements you intennd to perform with your protein...
Ion-Exchange chromatography or Iso-electric Focusing could do the trick as well, if you got the materials. Especially Iso-electric Focusing is very precise, up to a thousandth of pH accurate, if you did it right. Very few proteins have iso-electric points that close of a range. After IEF you could also put them on SDS-PAGE, just to be sure, and you have a 2D electroforesis
I agree with Masood, SDS is an easy method giving lot of surety regarding the purification of your enzyme. Well, Iso-electric focusing can further tell regarding the purity of the enzyme. In addition, Chromatography (Size Exclusion, ion exchange or reverse phase) and mass spectrometry can also be used.
Hope this will help
SDS PAGE should be done along side with homogeneity test (PAGE in the absence of SDS and beta mecaptoethanol) which will give you a distinct confirmation of what you are interested in
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