Oh, there is do much you can do! Lots of alternatives: 1. Decrease or increase the protein concentration (with a corresponding increase or decrease of precipitant. 2. Use a temperature gradient. 3. Set up in a capillary with liquid-liquid diffusion 4. SEED!
#3 or #4 are your best bets, probably. For the capillaries, using Hamilton syringes: Pipet 20 ul protein in a 50 ul capillary. Ease it away from the end. When you are done, make sure you can reach the liquid with the syringe from the other side. Hold your finger on one end. Seal the other end with sticky wax Layer 5 ul water on top. Carefully layer the precipitant (10-30 ul) on top of the water. You might want to use a 4mM glass capillary mounted on a syringe, instead of Hamiltons. Seal the other end. Wait 3 days. Then look. Naturally, you can scale this down using 20 ul capillaries, or even smaller X ray capillaries. But then you can't use syringes to load them, use smaller capillaries. On the upside, you could shoot directly from the experiment. This has worked well for me. Lisa -----Original Message----- From: CCP4 bulletin board on behalf of shivesh kumar Sent: Wed 8/22/2007 3:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal with precipitation Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar
