Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Jürgen Bosch
Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street,

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Nadir T. Mrabet
Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread David Briggs
Dear Jacob, I know that this is not the answer you were seeking, but for a modest increase in the amount of protein required, a couple of analytical ultracentrifugation experiments would be able to determine stoichiometry and binding affinity for such a system. AUC has the added benefit of being

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Jürgen Bosch
You don't necessarily need the second dimension. BN-PAGE gel: protein A alone| some proteins you know the size as reference|protein B alone| your mixture You will be able to see in the mixture a) one or b) multiple bands, since the Coomassie is equally distributed and attached to your protein

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Nadir T. Mrabet
Thanks Jürgen. Yes, you may show dissociation. However, especially if you deal with assembly, then it might be difficult, if not impossible, to tell your exact subunit composition if you runBNP only in the first direction. Jacob mentions the possible occurrence of complex assemblies (AB, BB,

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Hannes Uchtenhagen
Dear Jacob, somewhat adding to list of 'not really answering your question', here is the reference for native page that still uses a dye thereby trying to limit the influence of the charge on the speed. Might be helpful as they discuss some applications. Otherwise AUC really sounds like the

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Maia Cherney
That's interesting. Thanks. Maia Nadir T. Mrabet wrote: Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Sheemei Lok
Subject: Re: [ccp4bb] Native Gel Theory and Practice Here's just one example, which I quickly found from Reisinger and Eichacker. Isolation of membrane protein complexes by blue native electrophoresis. Methods Mol Biol (2008) vol. 424 pp. 423-31 Now Jacob has A 22 kDa B 17 kDa, the charge can

[ccp4bb] Native Gel Theory and Practice

2010-05-18 Thread Jacob Keller
Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-18 Thread Maia Cherney
Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration.

[ccp4bb] native gel

2009-02-12 Thread KN
Dear all, I am wondering if anyone is working on Blue Natve PAGE or other alternate methods. I need some help to troubleshoot my Native PAGE experiments. It will be great if anyone can help me in this regard. I am working on membrane proteins which have pI around less than 7. i need to run

Re: [ccp4bb] native gel

2009-02-12 Thread Zhijie Li
- From: KN kn...@auckland.ac.nz To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, February 12, 2009 10:30 PM Subject: [ccp4bb] native gel Dear all, I am wondering if anyone is working on Blue Natve PAGE or other alternate methods. I need some help to troubleshoot my Native PAGE experiments

Re: [ccp4bb] Native Gel Charge States Vs. Conformations Vs. Oligomeric States

2009-01-16 Thread Zhijie LI
Hello Jacob, The problem for native gel is that it is much more sensitive to a single charge difference than size differences. Also, the gel pattern may change greatly if you use a different buffer system. I had a case 2 years ago that my protein ran at 5 positions on a Laemmli(pH 8.8)