SDS PAGE - Validate method for MAb purity test (Apr/07/2004 )
I want to validate a purity analysis method using SDS PAGE. Can someone please provide me with guidance as to my next step? Is there a repository of method validation protocols which I can use as a template? The main band is around 70 KDa. We use a Invitrogen NuPAGE BIS -TRIS gel on a Mini-Gel.
Would SEC HPLC or CE be a better method for purity analysis?
If more info is required please let me know?
Purity analysis methods cannot be usually validated using SDS-PAGE. It is unfortunately open to criticism where a reviewer or an examiner can give you a hard time if they want to. Even if the gel is silver stained and single band homogeneity is demonstrated, questions of purity still exist. Have you considered mass fingerprinting?
Thank you for replying to my question. We have not looked at mass fingerprinting since we don't have the necessary equipment to perform the analysis.
If your protein is biologically active or has otherwise an activity that can be measured biologically, you can use the traditional validation method. This involves showing the progressive purification of your protein on a gel in parallel to increasing specific activity(Activity divided by protein added in assay).