Crystallization Trials

Materials

Milipore filter type HA 0.45 micron

Culture plates (Linbro model 76-033-05)

Protein in DDW or HEPES buffer (10 mg/ml)

Vacuum grease

Syringe with 18 G needle

Procedure

1.Millipore filter all solutions to be used.

2.Dialyze protein for 24 hours against water,if possible.Otherwise,include minimum requirements for keeping the protein soluble.If buffer is necessary use HEPES buffer.

3.For the initial screening use the buffers and precipitants recommended in the fast crystal screen (Jancarik,J.& Kim,S.H.1991 J.Appl.Cryst.24,409),as instructed,and the hanging drop method with the Linbro tissue culture plates that have 24 wells,1.7 cm diameter,1.6 cm deep.See diagram.

4.Use a protein solution with a starting concentration of 10 mg/ml.This concentration may have to be adjusted depending on the number of the drops that precipitate.The concentration is about correct when one-third to one-half of the drops develop precipitate within one week.

5.Deposit 0.7 ml of reservoir solution into the well.Combine 2 microliters of the protein solution and 2 microliters of the reservoir solution to make up the protein droplet.

6.Incubate one at room temperature and one at cold (4-10℃)temperature.

Notes

1.Crstallization screening kits and other supplies are available from Hampton Research.

Taken from methods by J.Jancarik and S.H.Kim

If you use methods from the Kinesin site,we ask that you cite the Kinesin Home Page and authors,or the appropriate source publication in your work.