PART B. Protein crystallization by sitting-drop vapor diffusion 1.) Now you are
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PART B. Protein crystallization by sitting-drop vapor diffusion 1.) Now you are ready to set up crystal trays. We will be using Cryschem plates, which rely on sitting-drop crystallization rather than the more common hanging-drop technique. Pipet a total of 1 mL of crystallization solution in each well of the plate, depending on the proportion of PEG you decide to use (100 pl buffer + X PL PEG + y pl ammonium sulfate + (900-(x+y)) pL Nanopure water). Mix the reservoir solution well by pipetting up and down and stirring with the pipet tip. -- NOTE: If you would like to just set up a well or two of lysozyme crystals, and if we have made the proper solutions, you are welcome to try the famous "15-minute crystals" recipe from Hampton Research. Of course, if you use this recipe you should use a microliter of concentrated lysozyme instead of your protein for those few wells The 15 Minute Lysozyme Crystals - By Enrico Stura Lysozyme: 100 mg/ml in 50 mm Sodium Acetate pH 4.5 Reservoir: 30% w/V MPEG 5,000, 1.0 M Sodium Chloride, 50 mM Sodium Acetate pH 4.5 Mix equal amount of lysozyme with reagent. Dilute protein and/or MPEG and/or Sodium Chloride for less nucleation, larger, and better shaped crystals. Batch or vapor diffusion method works fine. 2.) Place 1 pl of your concentrated protein solution in the dimple in the center of the plate, then mix by pulling it up and down with your pipette. Then use a different pipette to pick up 1 pl of crystallization solution from the well and add it to your protein drop, mixing gently by pipetting up and down. Avoid introducing air bubbles into the drop. Repeat this 23 times and you have a full tray set up. 3.) Carefully seal the tray with crystal clear tape over the top, and label with colored tape on the side. Move the tray very carefully to a storage place. You may store the tray at room temperature or in the cold room -- try to put it in a place out of direct sunlight and away from frequent vibrations or shaking. Always be careful when moving the tray.Explanation / Answer
No, the exact volume of the reservoir doesn't matter to the crystallization. 700 to 1000 microliters can be used. This large volume allows protein and precipitant mix well. Various factors affecting protein solubility can be added different solution with different pH, ionic strength, salts, precipitant s.
.etc
..can be added
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