Screening

Crystallization occurs when a substance (protein) is brought to supersaturating concentrations such that crystal nucleation and subsequent crystal growth occur. All new protein crystals must be screened for. Methods of screening include incomplete factorial (not so random sampling) and factorial (brute force: 100's to 1000's of solutions) screens. Crystallization trials are rapidly set up in Hampton 24-well plates. Each well is sealed with either a plastic or a siliconized-glass coverslip. Glass is used for crystallization solutions that have low surface tension (alcohols). All proteins prepared for crystallization must be PURE, monodisperse and concentrated to at least 10 mg/ml.

1. Label crystal trial plate
2. Remove any dust from plate with canned or filtered house-air
3. Apply vacuum grease to lip of each well using automatic grease machine or syringe
4. Pipette 0.65 ml of pre-made screen solution into well
5. Remove dust, with air, from coverslips and place on metal surface
6. Pipette 1 µl protein onto center of coverslip
7. Pipette 1 µl well solution (mother liquor) into protein drop
8. Invert coverslip and seal onto greased trial well, suspending protein drop over mother liquor
9. Continue until plate is finished
10. Immediately place plate in incubator (4, 16, 20, ... °C)

Two notes on temperature: 1) store most screening solutions at 4 °C, and 2) if the final crystallization temperature is 4 °C and you do not wish to set up trial in the cold room, firmly place trial plate on top of ice so that the bottom of the well contacts the ice during steps 4-9. Check plates at least once per week, but not too often because temperature variation may impede crystal growth.
 
 

Focused grid screening

Once crystals are obtained from screening. Prepare crystallization solutions for a focussed screen around the initial conditions. Primary variables are precipitant concentration, pH and protein concentration. All three dimensions can be tested on one plate if several protein drops of different concentration are suspended on one coverslip. Secondary variables include subtle differences in precipitant and buffer type and the presence of additives such as detergents or multivalent salts.