Stabilizing mother liquor is anything that (hopefully) will keep your seeds 'competent' (capable of nucleating single crystals). This may be anything but typically what people start with is something on the lines of the well solution supplemented perhaps with a bit extra precipitant.
As to when to add - this depends on how your drops behave. Specifically, if your drops can sit for weeks w/o nucleating then you may be able to add the seeds many days post-setup. However if you get showers of crystals in a few days (or hours) post setup then of course you should add seeds right away, or as soon as practical. The main goal is to have at least some of the seeds survive the transfer and hopefully nucleate big fat crystals for you. Incidentally if you just scrape a whisker or acupuncture needle across existing crystal(s) and then quickly sweep the end of it across fresh (or aged, whatever works) drops - you're likely to get seeds transferred. Passing the tip through several drops in series will create a pretty reliable dilution effect which I personally use quite often to seed - thus avoiding (most of the time) the need to mess with stabilizing solutions, crushed crystals and so forth. Proteases and crystallization - this is a hard guess to make. Assuming that what you see is indeed due to proteolysis and not some other effect (oxidation for instance, or dephosphorylation, or just the quirky physical chemistry of protein crystallization) then you could approach this in at least two ways: 1. try to figure out what sort of protease(s) is clipping your protein and where the cut sites are. Typical experiments involve analysis of gels, MS, selective inhibition of protease classes with chemical agents, etc. 2. just pick up a dozen or so different proteases and go to town on your protein - just make sure that you always set up a range of protein/protease ratios (start from perhaps 200:1 and up to 10,000:1 although sometimes 100:1 is also OK) Proteases to consider using in this case (by no means a complete list!) are: trypsin, chymotrypsin, papain, thermolysin, subtilisin, Arg-C, Glu-C, Lys-C, Asp-N, etc. In a pinch you can deliberately (gasp!) add a tiny amount of bacterial lysate to your protein. Just make sure to use 'cloning' grade cells rather than 'expression' strains which are commonly engineered to contain knockouts of OmpT, Lon, Prc and other proteases. Cheers, Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges _____ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joseph Brock Sent: Tuesday, September 15, 2009 7:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Seeding and protease questions Hi everyone, I have two conditions that I want to try and improve by microseeding. I have been reading the literature on the subject and am a bit confused as to what exactly is the "stabilizing mother liquor" that is used in the serial dilution of the seed stock. Is this just purified protein in its stabilizing buffer? Or a mixture of this and crystallization solution? I would also like to know what the general opinion is on WHEN to add these seed stocks to crystallization drop. Is addition generally more effective upon setup or after the drops have had a chance to equilibrate? Also, one of these conditions takes a LONG time to produce crystals (about 3 months). I understand this may be due to the slow digestion of my protein by low levels of co-purified proteases, whose slow nibbling eventually produce a truncated form that is more conducive to forming crystals. Thus, the process can be accelerated by the addition of something like pepsin to the crystallization drop. I was hoping that someone could offer advice as to what the optimal protease/concentration to use is such experiments? Many thanks in advance for the advice and everything I have learnt from this community in the past. Best regards, Joe PhD Student Research School of Chemistry Australian National University _____ Find out how here Get <http://windowslive.ninemsn.com.au/article.aspx?id=845706> Hotmail on your iPhone
