by Peter Nollert
March 23, 2010 20:00
Working with membrane proteins has its own joys. The main reason for this is that you're dealing with amphiphiles that show interesting behavior in solution. Trouble is, that such lipids and detergent are usually very expensive. Hence, micro methods that reduce the amount of detergent and membrane proteins for initial screening purposes are highly welcome. Daniel Gutmann et al. have devised rapid and cheap protocols to screen membrane protein samples for
- Solubilization in different detergents,
- Finding detergents for crystallization, and
- Finding the best buffer to combine detergents.
Gutmann, D.A.P. et al., (2007)
A high-throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay.
Protein Science, 16:1422-1428
Their main point is to replace size exclusion chromatography with ultracentrifugation. Running a sizing column usually requires multiple hundred mL of detergent solution, while spinning a protein sample in an ultracentrifuge tube and assaying the supernatant with SDS-PAGE can be done with 5 uL. That's a smart thing to do if you have access to 100,000 g and a suitable rotor holding small tubes. The protocol, glamorously called "ultracentrifugation dispersity sedimentation assay" is hence very similar to a conventional pull-down assay at high rotor speed. Ultracentrifugation is required to separate the non-sedimenting properly detergent solubilized membrane protein from heave aggregated membrane proteins.
I was glad to see that the authors showed images of the membrane protein crystals that they grew (using dodecyl maltoside as a detergent), together with diffraction images. But why on earth are they not disclosing the nature of the membrane protein? Can you imagine a paper describing in detail the "Expression and purification of membrane proteins" in the Materials and Methods section while disguising the nature of the three target proteins as MP-A, MP-B and MP-C? Well, MP stands for - you guessed it - membrane protein; some kind of ABC transporter. Here is it.
"I'm not telling you what I'm working on but I need the paper". Wiley InterScience, Protein Society & PROTEIN SCIENCE - how could you let this one slip through?
So much for the flak.
Other than that - great micro method contribution to help us do more with less.
Peter