X-tal protocols
Purity, homogeneity and stability of (membrane) proteins, as well as the individual properties of the protein, such as surface charge, isoelectric point, balance between hydrophilic and hydrophobic domains, are amongst the most important parameters for purification and crystallization.

This is why it is impossible to give a general protocol for the purification of proteins. But I can give a few guidelines:

The proper characterization of the protein sample requires:

Never mix batches from different purifications, due to small changes in the purity the optimal crystallization condition for each batch of protein can be different. If you don't purify the protein, ask for a copy of the purification protocol for every batch. Each batch optimally contains more than 10 mg of protein. If the protein can be stored safely at -20°C or -80°C, store the protein in aliquots to prevent multiple freeze-thaw cycles.

Purity of the protein sample.

(SDS page and IEF)

Picture of SDS Gel (illustration).

Different amounts of protein sample are loaded on the gel to check for contaminants and degradation products.

  1. Marker
  2. Large amount is loaded, contaminants are visible.
  3. Normal amount is loaded, protein looks pure.
  4. Low amount is loaded, degradation product is visible.

Crystals can occasionally be grown from impure samples, but size and quality can improve as the purity increases. If the protein contains contaminants and large amounts are available, further purification is recommended. But if the sample contains minor impurities and/or only a few milligrams of protein is available, try to find initial conditions for crystallization. Further purification can be used to optimize the crystals. Protein fragments by proteolysis can be a reason for failure or for crystals of a protein fragment. Change the protein purification (add protease inhibitor).

Sample homogeneity.

Avoid heterogeneity by optimizing the protein purification. If a purification protocol is changed, check the crystallization behavior by repeating the initial screen.

Protein concentration and starting buffer.

Dialysis tubes
Dialysis of small volumes (0.1-1 ml).

A piece of dialysis membrane (width 25 mm, length 50 mm) is soaked in water and cut to get a single layer of dialysis membrane (50 * 50 mm).
Pipet the sample in the Eppendorf tube (without lid) or other small tube.
Cover the tube with the dialysis membrane and place the O-ring (O-ring from dialysis button).
With a piece of parafilm the O-ring and membrane can be fixed.
Turn the tube upside down and check that the protein sample is in contact with the dialysis membrane.
With a piece of tape the dialysis set-up is placed in the buffer.

© 20-08-2018 Johan Zeelen