Validate SDS-Page (Collagen Typ I) - Looking for help about the point: specificity (Nov/02/2009 )
Iīm new at this forum and have a little problem with a validation.
I used the search-function and couldnīt find any thread with this topic.
I have to validate the sds-page at work.
We are characterizing collagen typ I most of the time.
I never did a validation before so I read a lot and learned what to do.
So I get the information to vaildate a few important things.
Robustness is one of the easy to describe parameters.
I have big trouble in phrasing "Specificity" and a little trouble with "limit of detection".
About limit of detection: Made a series of measurements with descending percentage of collagen. So I can see at which level I can detect the last visible bands in the gel. Ok. But I have hassle to validate "Specificity". SDS-PAGE isnīt specific to collagen typ I, how to show that?
My question: Has someone managed a validation for characterizing collagen typI with SDS-Page recently and is willing to help me with this?
Maybe this is a dumb question for some of you pros....
Thank you for reading and sorry for my english.
Hey K... to validate the specificity parameter in SDS PAGE means that you first show that it is wat u are looking for (Collagen). So run a standard along with it to match the migration. Next is run only the Sample buffer, diluent buffer etc to show that it do not give artifact or band at the same position where your protein or its dimer or any other related species gives. Thus you showed that your gel is now specific for your protein or interest..
Specificity covers within itself.. matrix interference (buffers run) and separation selectivity (in this you can show it can separate between dimer and monomer or alternatively any other HCP's give band at different position)
Also depends if you wanna use this test as just an identity test or a purity test!!!
any other opinions are welcome..
Thank you very much for your quick reply P.I.!
Good clear answer.
That helped a lot.
You re most welcome Kainer..
ya a great forum indeed!!!