Hello Ms Hardy,
Firstly, thank you for taking the time to reply and help. The two papers I mentioned in my first post were "Osmotic stress Induces Kanamycin Resistance in Escherichia coli
B23 through Increased Capsule formation - A. Kuzuhiyil, Y. Lee, A. Shim, A. Xiong, (2012); journal of experimental microbiology and immunology, vol. 6 5-10".
"Molecular Basis of Bacterial Outer Membrane Permeability Revisited - H. Nikaido, (2003); Microbiology and molecular biology reviews, vol. 67(4)"
However, neither of these contains methods/experimentals on porin purification, there just where I got the basic idea for my project. I do have about 15ish papers with protocols in, which I've been using to construct my own protocol; GE healthcare also have a booklet available as a downloadable pdf file, the subject of which is isolation of membrane proteins. It's called purifying challenging proteins.
I checked today, and unfortunately the lab I'm using doesn't have a amicon ultrafiltration membrane, but I do have access to spin filters which are "apparently similar" although I don't think I'd have time to figure out which unit I would need to use. I think? they were called 'Bio-Rad spin filter tube/columns' they use a centrifuge to force the homogenate through a membrane (again I think
). Although, would it not be possible to collect the outer membrane fraction through differential centrifugation at 100,000g and then use a detergent such as Triton X-100 or tween to solubilise the protein components of the cell membrane; and then collect the OmpC and other approximate proteins using a density gradient centrifugation step?
Other than the ultrafilter, I have most of the other materials. I have as much media as I need and culture flasks, autoclave, ddH2O and an incubator. The media I'm using is a custom broth, based on a lysogeny broth recipe (from this site - http://www.bioprotocols.info/model_orga ... plates.php
), only I've tweaked the ingredients to increase the osmolarity and pH. Per litre it contains 1g of lemoco lab powder from Oxoid, 2g of yeast extract, 2g peptone and 3g tryptone, it also contains 0.8 NaCl and 0.2 glucose which raises the osmol's by 3.0.
As for the buffers I'm pretty sure we have Tris-HCl, although could I not use Tris-buffered saline or even phosphate buffered saline???? I also have access to MgCl2 and (I think) to SDS, which is sodium dodecyl sulfate, right??? The EDTA and saline won't be a problem either, and we have SDS PAGE electrophoresis equipment and reagents. I don't have access to a sonificator, although I imagine I can use a pressure cell or lysozyme, do you think this will matter? Also, I don't think I can get the 2-mercaptoethanol. At the moment this is what I have and don't have. Oh and I have access to low and medium pressure chromatography systems, specifically the Buchi sepacore, which has a pressure range from 10-50 bar, and can take a litre of sample at maximum. Or I have access to bio-rad econo-glass columns which I could pack with media and then attach to either a low pressure air pump or apply a mild vacuum via a Buchner flask??? also I need to order protease and phosphatase Inhibitors, so I could really use some advice on which ones I absolutely need or if I can get away with just keeping the homogenate cold (for example).
essentially I want to do the best I can which the budget I have, I can access all of the reagents, like the EDTA and buffers, and the equipment free of charge; and possibly the size exclusion media. However anything that I need to order-in I'll have to pay for, so I'm open to improvisation.
I've now also got the K-12 cells from 4 litres of culture, I pelleted them and then re-suspended them in a PBS with 10% glycerol (and 10% cow serum?) then flash froze them with liquid nitrogen. Although most of the protocols that I've read on-line say to store the cells at -20C > -80C , but as I would have to disturb people to get them in and out of storage at that temperature I've put them in a normal freezer, one of the lab technicians said that would be ok in the short term, once I've lysed them I'll store them at -80C. Do you think that will be alright?
I really appreciate your help with this. Also I'll make a list of the most important sources I'm using and post them soon (most likely tonight or tomorrow)