Almost every purification we can think of begins with a muckfarming step, this involves some initial attempt to remove that protein from it natural biological mileau. Almost all begin in a horrifically impure state, that's why all the PMSF, protease inhibitors, RNA inhibitors, DNAse inhibitors. This is often the single most important step in any purification. The spectrum of contaminants within the natural mileau are often the greatest threat to a successful purification, however, these contaminants are magnitudes more common in the first extraction step than on most desktops.
Hardy proteins like antibodies can be recovered from unplanned accidents, other proteins may undergo oxidation or modification altering their properties. Proteins run the spectrum of stability, from Botulinum neurotoxins that are sensitive to heat and oxidation to ribonucleases that can be abused in a great variety of ways and still function. And BTW if you are dropping botox, you have bigger problems. I once watched an mAb antibody purification spill into a drawer because of a mechanical glitch in a Gilson microfractionator, for some reason the cellulose-based lining of the drawer had become hydrophobic and thus did not absorb the solvent. (The drawer had be cleared to hold the fractionator but was not cleaned) The material was carefully removed and spun at 30,000 g. After which point it underwent both purity and binding analysis. Both were excellent, repurifying a small sample did not improve the purity. [Since this was being used as a pilot experiment we did utilize the material] Antibodies have a half-life in serum of 120 days (37'C) and are continually being exposed to serum oxidants. Thus they are not particularly heat or oxygen sensitive. The major component of contamination is dust, this is composed of cellulose, saliva proteins, skin proteins, and arthropod debris (dust mites, feces, etc). There may also be residue of cleaning agents. Most of this material is insoluble, that is why dust needs a little bit of surfactant to (e.g. hand or dishwashing soap) to wet-wipe. These may interact with proteins. Many of these components will clump in water and spin-clarify. Situations to watch out for in pelleting are desired structures that have very long axial ratios as these can become trapped by the Augston-Johnson [sp] effect (whereby very long strands become entangled in falling strands and separate slowly, and require longer times to effectively differentially sediment). Proteins with high solubility and low axial ratios should separate effectively from these components on spin down, further separation can be achieved by underlaying a small amount of 8% glycerol at the bottom of the tube (swinging bucket rotor t! ype with proper deceleration). This means the last few mls in the tube will be avoided. Another very effective way for exchanging fluids is using the a desalting column, such as G15, this can quickly get rid of the solutes of low molecular weight (bug piss, human sweat, saliva solutes). G15 purifications do not have to be precise, OD280 can be used to monitor the progress of the protein, G25 coarse can be used for higher molecular weight proteins. Basically, a properly equilibrated G15/25 column allows you to rapidly replace a corrupted solvent with the proper solvent. When your activity/OD280 peak drops, you discard that which follows. Thus all that you have left are high-sed soluble material that has a radius over a certain size. This capability means that you can treat/preserve some proteins with inhibitors if they are momentarily exposed and then buy some time to set up a filter-top desalting column. G15/25 do not have to be long (only 6 inches will do, and they can be quite wide in diameter, which means you can get a good capacity, and they can be recycled! as long as you prefilter or spin the solute). So in a pinch these can clean things up fast. The final issue is political, you really don't want to write this in your material and methods section. So if this is going into a final data for a paper, use a protocol that is actually going to be reproduced by the reader. There may be some factor on your desktop that causes your protein to double its activity [e.g. Formula 409 residue, partially burnt PCB-laced cannibus fragments from that last "lab" party that the chairman never heard about, chili sauce from the janitor's super-spicey carne al-pastor taco (the one he added extra-acidic habernero sauce to), your double-moca 'glucose surprise' cuppachino (the one with the BP-gulf oil slick floating over the rim of the cup)], and some grad student might be cursing you for 2 years in his thesis program trying to reproduce your results. -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Phelan, Paul J. Sent: Sunday, December 18, 2011 4:06 PM To: [email protected] Subject: Can spilled protein be recovered? This is kind of a trivial question, less technical than the usual, but I'm just wondering what people's thoughts are on whether it is worth trying to recover spilled protein. I recently finished purifying some TEV protease which I needed for another purification, and I had 62 mg of it in solution in a 50 ml Falcon tube, until I dropped it on the bench. All I could do was watch it spread out across the bench, although I considered trying to suck it up or soak it up in something absorbent to recover it, but I was advised to just "let it go". It's not a difficult purification, so I can repeat it, but has anyone spilled important protein and successfully recovered it, using some clever methods? Sincerely, Paul Phelan Tufts University Boston _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
