Preparing protein Crystals
Protein Crystallization Tutorials: http://xray.bmc.uu.se/terese/crystallization/tutorials/tutorial1.html
How to prepare protein Crystals
First, do the calculation how much protein is needed for screens. You would need 4-5 plates for Hampton research screen 1& 2, probably 4-5 plates for index screen. In addition to that you want to do finer screen which will take about 2-5 plates depending on how lucky you are.
Each screen you do 2-4 drops and each drops need to contain 1-2 uL of 16-24mg/mL protein. That is ~20ug/uL x1 (or 2) uL x 4 (or 2) drops x 12 plates x 24 wells/plate=23 mg protein.
A 2-liter prep should be enough which typically yield more than 20mg of protein in our lab.
1. Protein solution.
20mM MES buffer pH 6.0, (decalcified if using calcium binding protein).
Do a buffer exchange in concentrator for 4~5 repeats and eventually to a concentration of about 25~35mg/mL because the next step will dilute the protein (Measure protein concentration with nano drop, remember to use the flow through to blank and re-blank).
Run the protein through spin column, if you have a volume of about 1~1.5mL then use PD-10 column GE healthcare. If the volume is less than 0.75mL use the Miditrap G25 column. If it is between 0.75mL and 1mL I guess both are fine. Measure the protein concentration after the spin column. At the final spin give it sometime to settle and incubate it with your ligand. (metal, substrate etc. remember when you add metal you are further diluting the concentration of protein unless you have a super concentrated ligand stock or large volume of protein solution)
2. Spotting crystal (hanging drop method)
Pre-incubate all solution and pipette tips, in the constant temperature crystal room for at least 1 hour to equilibrate to the room temperature.
Add 1mL screen solution to the bottom of the 24 well plate and this is call base solution. (Make sure you label all wells before you do this step.)
Get 1 glass slip place/invert (depend on which side was exposed to air) it on the lid of the plate. Draw 1uL of base solution from the well and place close to the center the slip and take 1 drop from your protein pipette mix with the base solution drop 3~4 times. You can spot 2uL to 2uL as well. You can spot multiple drops.
Invert the slip on the well, press very gently on the glass against the silica gel. Use a pair of tweezers’ back to press on the edge down so that the slip is air tight against the well. This step is important.
If the slip cracks do not panic, the drop can be saved. Just peel off the slip and transfer the drop (s) to a new one. There might not be enough gel on the well (we do not have additional gel but you can either use the leftover gel on the well, turn the slip while pressing down, or scrap some from the old slip). Don’t waste that well. But do not take from other wells, because you do not want to ruin other wells to save this one.
3. Taking hits
If you find hits, you need to use a condition screen with gradients to further screen for better crystals.
Basically decide which PEG is best i.e. large molecular weight versus smaller molecular weight. Screen for pH, PEG concentration first.
Then probably screen for Salt concentration, temperature.
Nov. 13, 2012 We finally obtained crystal for our PKC alpha C2 domain in Combination with Cadmium ions.
Left: Shperical Crystal, Middle: Tube crystal, Right: Salt crystal
These morhorlogies on the left and middle pictures are not usually obsereved. We think it is likely due to protein self assembly. But at this stage, we are just waiting to get some x-ray patterns off of them!
In General HOW TO Distinguish between Protein crystals and salt crystals?
1. Look at color, salt crystals typically are small blobs, or small treads or sponge like.
2.Use a microscope with polarizer and look at the crystal. In layman’s terms a salt crystal is more colorful.
3. It is generally said that protein crystals have sharp edges. It is not true cause salt crystals can have sharp edges such as cesium chloride. But if you do a screen and you have similar morhorlogies under different salt conditions, you should be pretty sure that it is protein provided it looks nothing like described above.
4.Protein crystals have a larger water content, they tend to crack. So if your crystal crack a lot, it probably is protein.
Experiment confirmed that the straw shaped crystal being protein. They actually diffract quite nicely. The speherical crystal did not diffract.
Structure of the crystal was solved, with a resolution of 1.74A.
This work is supported by the Welch Foundation.
Zheng Long at
Department of Biochemistry & Biophysics
Texas A&M University