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Crystallography for beginners – part 4 – setting up your first crystallization experiment

You have purified your protein and you’re ready for your first protein crystallization experiment. In this post I will give you step by step instructions how to set your first protein crystallization experiment.

Materials and equipment list

Before you begin, verify you have the resources required for protein crystallization experiment:

  • Temperature and humid-controlled refrigerator/chamber/room for crystallization plate incubation. Make sure you have them set to 20°C and 4°C (optional).
  • Your protein at concentration of ~300-600µM (10-20 mg/ml for a 30 kDa protein).
  • A commercial sparse-matrix screening kit such as is sold by Hampton Research, Molecular dimensions, Emerald biosciences etc. Order deep well blockand transfer each condition of the matrix to a new well in the block. Seal and keep at 4°C.
    • Looking at the lists of kits, you must ask yourself which one to order? Well, you’ll want the screen which will sparsely sample as much as possible different crystallizing precipitant (salts, organic and inorganic solvents, PEGS etc.) while varying the conditions slightly for every type of precipitant. Some names that we have been using in the lab: Index, Crystal screen 1+2, Wizard, JCSG, structure etc.
  • 96-well sitting drop crystallization plate (MRC) such as this one.
  • A well calibrated set of 0.1-2µl, 0.5-10µl and 10-200µl pipettors + tips.
  • A multichannel of 10-100.
  • Crystallography sealing tape such as here and regular standard width office tape such as 3M of Scotch (I personally find the mate finish of the 3M tape to be just at the right binding capabilities, not too light and not too strong)

Setting up thecrystallization plate

1. Take a new 96-well sitting drop plate and write on its side the following information:

– Date
– Your name (and lab if the incubator serves other labs as well).
– Protein name and concentration at the upper and lower wells (use “up” and “down” arrows).
– Buffer composition of the protein solution.
– Name of the sparse matrix.
– Incubation temperature.

2. Prepare your protein solution at x1 and at x0.5 concentrations.
3. Using the multichannel, copy 50-80 micolitters of the matrix stock onto the narrow well of each column/row (“reservoir well”).
4. Using an aluminum foil cover the plate except for column 1.
5. Use the 0.1-2 microlitter pipette to draw half a microlitter from the lower concentration protein solution and dispense it onto the top well.
–  To avoid inserting air bubbles push the pipette button to the its first stopping point.
6. With the same tip draw half a microlitter from the higher concentration solution and place it in the lower well.
7. Replace the used tip with a new one and draw half a microlitter from the reservoir well and dispense it without mixing onto the upper drop. Do the same for the lower well.
8. Repeat the same operation for all rows B-H; do it as fast as you can since the experiment of the dispensed drops is running and the open air dehydrates the water off the drops.
9. Cut off a plate-width strip of the office tape and carefully seal the first column.
10. Repeat this step for all the rest of the column.
11. When finishing, remove the office tape and quickly as possible re-seal the plate with crystallography-grade sealing tape. Make sure the seal is complete along the rims of the wells and that no air can pass between the wells.
12. Place the plate at the incubator and monitor for crystal growth. How do you do that? See my next post on monitoring and evaluation of crystallization experiments.

Searching for the SDS-PAGE image of your protein? You might want to consider Labguru, a web-based laboratory management system. Go ahead, it’s free!

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