Hi Careina, Gels certainly are convenient and powerful tools when they work. For normal native gel, do not forget that if you are using the Laemmlli buffer system (ie. tris-glycine), the actual pH during running is pH 9 and the ionic strength changes over time. There are other buffer systems, but you still need to be careful with the results. The other consideration is that when running a dimer in gel, if the dimer is not very stable, during a long run (for example, regular native gel often take hours), the dimer can dissociate significantly, resulting in a smear or total loss of the dimer band.
I think if you can run gel-filtration experiments(a little more expensive in terms of equipment and protein consumption), you probably can draw conclusions with more confidence due to: 1) you can see the peaks in 20 minutes so even if the dimmer dissociates it will dissociate less in less time; 2) you can equilibrate the column in your buffers so there is no issue about the real condition you are testing. The drawback is that you can't test your different conditions in parallel. But with gels, your conditions only exist in the loading wells, when the proteins enter the gel and spend the 1-5 hrs there, your initial conditions are lost anyways. Zhijie From: Careina Edgooms Sent: Thursday, March 28, 2013 3:03 AM To: Zhijie Li Subject: Re: [ccp4bb] off topic, BN PAGE Hi Zhijie Thanks for the helpful reply. The attractive thing about BN PAGE (if it works of course) is that it is so quick, inexpensive and simple to perform. I am working with a protein that exists in the native state as a mixture of monomer and dimer. I wish to monitor the effects of various things on this monomer dimer equilibrium. The simplest way of doing this is to run a gel at the end of the experiment and quantify the bands with densitometry to check the effect on the monomer dimer equilibrium. When I run this protein on a BN PAGE gel it runs as 2 bands. I initially assumed these 2 bands to be a true representation of the monomer dimer equilibrium but now I am wondering if the coomassie could actually interfere with the contacts so that even if the protein was in a state where it was entirely dimeric, it would appear on the gel as 2 bands because the coomassie is interfering with the dimer interface. What I'm attempting to do now is run a regular native page without coomassie. Because my protein is very basic this means I will have to swap the electrodes. If this also shows 2 bands then I wonder if it is safe to assume that the BN PAGE works and coomassie does not interfere? Careina -------------------------------------------------------------------------------- From: Zhijie Li <zhijie...@utoronto.ca> To: Careina Edgooms <careinaedgo...@yahoo.com> Cc: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, March 28, 2013 1:28 AM Subject: Re: [ccp4bb] off topic, BN PAGE Hi Careina, BN PAGE can be affected by many factors. Considering the complexity and the chance of winning and the amount of information you gain even when you win, I do not recommend fighting it. BN PAGE, like other gel-based methods, requires that your complex is fairly stable - not having a weak affinity and not a high dissociation rate. Also the complex formation should be compatible with the gel running condition: the pH and salts and other things. Coomassie itself may compete for some hydrophobic surfaces or positively charged residues on the proteins - in such case there's little you can do. If you see a band corresponding to the expected complex weight, then congratulations, BN gel might be your tool (with cautions though as you can also get false positives with BN gel). But if not, then my suggestion is to move to other techniques such as gel filtration, analytical ultracentrifugation, BiaCore, ITC and so on. I had a case in which I could confidently show complex formation with gel-filtration and Biacore, but not with BN gel. Zhijie From: Careina Edgooms Sent: Wednesday, March 27, 2013 5:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic, BN PAGE Hi Has anyone found the coomassie in a BN PAGE to be interfering with the oligomeric structure of their protein? If so, how did you deal with this? Thanks Careina
