= My Thinking Throughout, of course, we use electrophoresis grade reagents and milliQ (or better, HPLC_ grade water. == Protein Extraction Need to: - Minimise degradation -- Proteases -- Phosphatases - Minimise losses -- Especially hydrophobic proteins - Halt biological processes -- Depends on the experiment; for mapping, not important, for studying transcriptional responses, a few minutes doesn't matter, for studying signalling, every second counts So: - Go from live, happy cells to dead, stopped cells -- Snap-freeze suspended cells in L__N2, then melt by sonication into denaturant w/ inhibitors -- Wash + dry attatched cells (rinse w/P__B__S + aspirate), then cover in denaturant w/ inhibitors + scrape (+ sonicate?) --- It has been suggested that there is too much salt in P__B__S, which can interfere with I__E__F; phosphate- or tris-buffered sucrose could be used instead -- The classic denaturant is T__C__A in acetone; T__C__A in ethoxyethane is probably better - Precipitate at low temp, then spin and aspirate precipitant, leaving a dry, dead raw protein pellet == Sample Preparation Need to: - Block cysteines - Remove salts (make sample too conductive) - Remove lipids (interfere w/I__E__F) Note that if precipitation will be used, detergents and chaotropes must be avoided. So: - Take dry pellet, resuspend in a generic buffer (50 mM tris pH 8.0, salt, etc) with D__T__T, boil, add iodoacetamide in the dark, quench with D__T__T; this leaves the proteins with blocked cysteines in a very salty buffer, with plenty of contaminants (salts, lipids, etc); precipitate with T__C__A to get back to a dry pellet -- If there was some additive which would improve protein solubility here without compromising later precipitation, it would be most helpful - Do N (2?) rounds of desalting by suspending in water, then precipitating with T__C__A and centrifugation (at least one round will be needed, because of the residue from the blocking) - Do N (2?) rounds of delipidation by suspending in ethoxyethane, then precipitating and centrifugation (at least one round will be needed, because of the T__C__A); ethoxyethane is supposedly better than acetone for this (hydrophobic proteins are less soluble in it) - Resuspend in sample buffer; this requires ampholytes (should be present at resuspension, for buffering), thiourea/urea (thiourea is generally better, but is not compatible with some of the better detergents), detergents (see below) and glycerol or isopropanol (to reduce electroosmosic drift; glycerol is probably the better of the two); note that it does not include reductants (as the cysteines are blocked) or dye, both of which are charged and so contribute to I__E__F heating -- Detergents; these are well known, but the key is to use a cocktail, as a mix of detergents gives a much better result than just using one; the basis for the mix should be C__H__A__P__S or C__H__A__P__S__O, with an N__D__S__B being a wonderful additive (as it has no limiting concentration!), A__S__B-14 being good for membrane proteins, S__B3-10 being a better equivalent of C__H__A__P__S, the M__E__G__A and Brij being dark horses and N__P-40, Triton, Tween and octylglucoside (or other alkylglucosides) being cheap and cheeful extras --- See http://users.ox.ac.uk/~univ0938/bioch/surfactants.html -- Don't heat this above 30C; the urea will degrade Resuspension is generally best done by sonication on ice. External sonication is better than internal, as there is no needle in contact with the sample. == I__E__F - Reswell under voltage (100V) - Load sample in reswell buffer, except for 6-11, when cup loading at anode (?) may be better - Use full power (8 kV good, 10 kV better) - Run at 4C; do everything in the cold room -- There is some suggestion that going below 20C is not good if urea is used; so don't use urea == S__D__S-P__A__G__E This is pretty standardised. Running at 4C rather than 14C might be good. The necessity of urea in the second dimension is also not clear. Thinner gels and higher voltages are better, as always. The only other idea i have is that rather than equilibrating the strips by soaking in buffer (and thereby risking loss or diffusion of proteins), they should be loaded as-is (after removing oil), and a big load of SDS loaded on top (a strong solution of SDS in buffer, with either sucrose or glycerol for density); the SDS would electrophorese down into the strips and pick up the proteins there. You never know, it might work! == Imaging The best generally available stain is Sypro Ruby; M__P17 is better, but secret. Silver or gold are okay, but not as convenient or uniform as Sypro, and no more sensitive. Standard protocols for staining and scanning are perfectly good. Destaining should be used. The scanner should be persuaded not to do any image processing whatsoever. A radical idea would be to use UV spectrophotometry to quantitate proteins without staining, but this might not be sensitive enough, and might be impossible due to absorbtion from gel or buffer components. == Analysis Median filter (or something smarter, like non-parametrically checking each pixel against its neighbours, and replacing outliers with the mean/median/interpolation of neighbours) can be used to eliminate shot noise. Rolling ball and rolling disc should be used to eliminate background and streaking. - About shot noise elimination. Median filter is too rough, especially given rare noise (check that the noise is indeed rare -- ed.). The idea would be to judge a pixel by taking its 8 neighbours, sorting their values, discarding the top and bottom (in case those are also noise), then defining a plausible range for the central pixel as being something like three times the range of the six remaining neighbour values (say these are 110-130, the plausible range would be 90-150). If the pixel under test was outside the range, it would be replaced with some interpolation between the neighbours (eg a mean or median). The number of pixels to discard and the multiplier for the plausible range are parameters that need playing with. If there is a standard statistical test for this sort of thing (it would probably be nonparametric), it would be better. The rest is a bit of a mystery. = Web Links - http://www.queens-pfd.ca/images/pdfs/2DE%20outline.pdf - http://www.weihenstephan.de/blm/deg/ = Papers This is all of Proteomics 2003, up to No 6. Brian A. Stanley, Irina Neverova, Heather A. Brown, Jennifer E. Van Eyk Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium Maurizio Bruschi, Luca Musante, Giovanni Candiano, Gian Marco Ghiggeri, Ben Herbert, Francesca Antonucci, Pier Giorgio Righetti Soft immobilized pH gradient gels in proteome analysis: A follow-up Mike Rogers, Jim Graham, Robert P. Tonge Using statistical image models for objective evaluation of spot detection in two-dimensional gels Mike Rogers, Jim Graham, Robert P. Tonge Statistical models of shape for the analysis of protein spots in two-dimensional electrophoresis gel images Paul Cutler, Geoffrey Heald, Ian R. White, Jason Ruan A novel approach to spot detection for two-dimensional gel electrophoresis images using pixel value collection Sylvie Luche, Véronique Santoni, Thierry Rabilloud Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis Christophe Tastet, Stéphane Charmont, Mireille Chevallet, Sylvie Luche, Thierry Rabilloud Structure-efficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis Read proteomics 2002 and earlier. CategoryBiology CategoryLaboratory