Crystal Handling Kit
For in vitro use only!
Shipping: shipped at ambient temperature
Storage Conditions: store at 4 °C
Shelf Life: 12 months
The Crystal Handling Kit is designed as a tool to practice protein crystallization, crystal mounting and X-ray data collection.
- Lysozyme (chicken egg white) dissolved in water, 2 aliquots, 250 μl (20 mg/ml) each
- Proteinase K (Tritirachium album) lyophilised, 2 aliquots, 5 mg each
- Xylanase (Trichoderma longibrachiatum) dissolved in 43 % v/v Glycerol, 180 mM Na/K phoshate buffer, pH 7.0
2 aliquots, 150 μl (34 mg/ml) each
- Solubilization Buffer, 2 aliquots of 0.5 ml per protein (see Table 1)
- Crystallization Buffer 100 ml per protein (see Table 1)
- Dual-Thickness MicroMount™ Assortment A2,
- 3 Goniometer Bases, Cat.-No. GB-B5
Table 1: Lysozyme, Proteinase K and Xylanase and their respective solubilization and crystallization buffers.
|Protein || Solubilization Buffer || Crystallization Buffer|
|Lysozyme || 100 mM Sodium Acetate, pH 4.0 || 1.5 M Sodium Chloride,|
50 mM Sodium Acetate, pH 4.0
|Proteinase K || 100 mM HEPES, pH 7.0 || 1.2 M Ammonium Sulfate,|
100 mM Tris-HCl, pH 8.0
|Xylanase || 180 mM Na/K phosphate buffer, pH 7.0 || 0.8 M Na/K phosphate buffer, pH 7.5|
Crystallization of the model proteins
The following protocol describes crystallization using the hanging drop vapor diffusion method. In addition to the kit you require hanging drop plates, (e.g. SuperClear™ Plates), cover slides and silicone grease.
- For Proteinase K dissolve 1 aliquot of 5 mg protein in 0.5 ml solubilization buffer in order to obtain a protein solution with a final concentration of 10 mg/ml. Mix protein and solubilization buffer very gently to prevent denaturation of the sample.
Lysozyme and Xylanase are already dissolved. Use the appropriate solubilization buffer to dissolve to 10 mg/ml.
Alternatively you can prepare serial dilutions of the stock solutions with solubilization buffer. Please note, that the xylanase drops will then have varying glycerol concentration depending on the dilution.
- Grease the crystallization plate by applying silicone grease evenly to the circular brim around the individual reservoirs.
- Pipette 1 ml of the respective crystallization buffer into each reservoir well of a hanging-drop plate.
- Pipette 2 μl of the freshly made protein solution onto the center of the cover slide.
- Carefully add 2 μl of the reservoir solution to the sample drop, without forming bubbles in the drop
- Invert the cover slide and place it over the reservoir so that the hanging drop is positioned over the center of the reservoir.
- Repeat steps 4 through 6 for the remaining reservoirs.
- Incubate and store the crystallization plate at 4°C, and minimize plate vibrations.
- Examine the crystallization plate under a microscope daily. Crystals should be observed after 2-10 days.
Crystal Mounting using MiTeGen’s MicroMounts™
- Select a MicroMount with a crystal aperture matched to the size of your crystal. Use an aperture that is slightly smaller than your crystal, so that the crystal rests on the edge of the aperture. This will minimize the amount of surrounding liquid and reduce your background X-ray scatter.
- Secure the mount in a goniometer base ('cap') using a small amount of high vacuum grease (Dow-Corning #976V), Duco Cement or epoxy. The base can be held using a magnetic wand. Alternatively, insert the mount into a 0.7 mm mechanical pencil.
- Slowly insert the mount tip into the crystal-containing drop to minimize fluid motion. Center the crystal over the crystal aperture and then slowly lift the mount while allowing the crystal to sediment towards it.
- For crystals that have settled to the bottom of a crystallization plate, try sliding the mount tip along the bottom of the plate and under your crystal. If the crystal has adhered to the plate, gently push on it using the tip of the mount.
- If there is still significant excess liquid around your sample and you're using MicroMounts, you can insert a size 15 or XF paper wick into the wicking hole to absorb excess fluid. Work slowly to ensure the crystal stays in place.
- Flash cool the crystal by your favorite method. The simple hyperquenching protocol of Warkentin et al.  gives much faster cooling and allows almost all crystals to be successfully cooled without using penetrating cryoprotectants.
For crystals smaller than 50 μm:
- Use LV CryoOil or work in a humidified environment or a cold room to prevent dehydration.
- Allow enough time for the crystal to sediment onto the mount, and remove the mount from the drop very slowly. Smaller crystals take much longer to sediment.
- Try Mitegen's MicroMesh mounts to sift out small crystals.
Please visit the Technical Notes section at www.mitegen.com for more detailed tips on using MiTeGen's products for mounting small crystals, for mounting needles, rods and plates, for minimizing background X-ray scatter and maximizing resolution, and for minimizing sample motion in the cold gas stream.
- Lysozyme, #CO-401
- Xylanase, #CO-403
- Proteinase K, #CO-404
- SuperClear™ Plates, 24 well, #CPL-130
- SuperClear™ Plates, 24 well, pregreased, #CPL-132
- Cover Slides, ø 22 mm, 0.5 mm thick, #CSL-107
- Bayer Silicone Grease, medium viscosity, #CGR-101
- DT MicroMount™ Small Aperture Assortment, #M2-L18SP-A1
- DT MicroMount™ Medium Aperture Assortment, #M2-L18SP-A2
- MicroMount™ Large Aperture Assortment, #M1-L18SP-A3
- MicroMesh™ Mounts - Assortment, #M3-L18SP-A1
- SPINE standard Goniometer Bases, #GB-B5-10
- W-15 Liquid Wicks, #W-15
- W-XF Liquid Wicks, #W-XF
- LV Cryo Oil, #LVCO-1
 Warkentin et al. (2006) Hyperquenching for protein cryocrystallography. J. Appl. Cryst. 39: 805.
Availability Restriction: nonsaleable to USA and Japan