Hampton Research

RAMC 1997 Crystallization Tips

Organized by Bob Cudney and Allan D'Arcy
Location: Le Bischenberg, France

Filtering the protein

To reduce the number of crystals and increase their size, try filtering the protein solution prior to setting up the experiment. Try the following filter sizes: 0.22, 0.1 micron and 300 kD molecular weight cut-off. Try the Millipore centrifugal filters.

Naomi Chayen

Pseudo-Microseeding

When working with crystals that grow fairly rapidly (one day) try the following. Pipette multiple protein drops (2 to 4 works best) onto the cover slide. Using a single pipette tip, get the reservoir solution to mix with the first drop. Now, go back to into the reservoir with the same tip to get the reservoir solution for the second drop. Continue for the remaining drops with the same tip. In certain cases, seeding starts very quickly, so by using the same tip one can introduce minute seeds to successive drops. Use the same cover slide with multiple drops to minimize evaporation.

Mike Sintchak

Low molecular weight PEGs

When screening with low molecular weight PEGs try microbatch. Crystals appear rapidly with PEG 400-2000. To convert to vapor diffusion use 0.2 M buffer in the well and a 1:1 drop ratio. Try using a positive displacement pipette such as the Anachem Microman 1 - 10 microliter. These are much more accurate.

Lesley Haire

Purest is not the best

A protein which was purified and showed some faint bands of contaminants on a native gel was crystallized and solved successfully. The same protein, purified by HPLC and resulting in a single band native gel did not crystallize.

Michal Harel

Cryoprotectant

When making up cryoprotectant solutions containing glycerol, put a test tube of glycerol in a beaker of warm water. The viscosity falls and it is easier to pipette accurately.

Elspeth Garman

Mass spectrometry

We found mass spectrometry like ESMS and MALDI highly efficient in determining impurities and/or microheterogeneities in our protein sample/batch. In most cases it is a simple, straight forward method which requires a minimum amount of sample. In some cases it has shown to detect impurities/microheterogeneities when other techniques did not.

David Leys