Product in the spotlight

---

Lysozyme Crystallization Procedure

Chemicals needed:

• Sigma Lysozyme from Chicken Egg White #L-6876
• Sodium Chloride
• Sodium Acetate buffer
• Sodium Azide
• Distilled water

Procedure:

1. Prepare 75mg/ml sample of Lysozyme in 0.1 M NaAc pH 4.8 plus 0.02% (w/v) Sodium azide.
2. Linbro or VDX tray well solution consists of 1ml of 6.5% (w/v) NaCl, 0.1 M NaAc pH 4.8, and 0.02% (w/v) Sodium azide.
3. On a clean silated cover slip create a drop of 5, 6, or 7 microliters Lysozyme solution, and 5, 4, or 3 microliters of well solution, respectfully, for a total of 10 microliters.
4. Seal a cover slip with grease over each well.
5. Crystals should grow in about 1-2 days.

May vary drop ratio between protein and well, or vary Lysozyme concentration between 50mg/ml and 75mg/ml, if crystal size is too small.

Approximate cell parameters for Tetragonal lysozyme:
Unfrozen: about 79.2 x 79.2 x 37.9
Frozen: about 78.8 x 78.8 x 36.9

---

Lysozyme crystallization procedure for low temp use

Chemicals needed:

• Sigma Lysozyme from Chicken Egg White #L-6876
• Sodium Chloride
• Sodium Acetate buffer
• Ethylene Glycol
• Sodium Azide
• Distilled water

Procedure:

1. Prepare 75mg/ml sample of Lysozyme in 0.1 M NaAc pH 4.8 plus 0.02% (w/v) Sodium azide.
2. Linbro or VDX tray well solution consists of 1ml of 10% (w/v) NaCl, 0.1M NaAc pH 4.8, 0.02% (w/v) Sodium azide, and 25% (v/v) Ethylene Glycol.
3. On a clean silated cover slip create a drop of 6 or 7 microliters Lysozyme solution, and 4 or 3 microliters of well solution, respectfully, for a total drop size of 10 microliters.
4. Seal a cover slip with grease over each well.
5. Crystals should grow in about 1-2 days and be ready for freezing.

If crystals are smaller than desired try varying drop ratio between protein and well, or vary Lysozyme concentration between 50mg/ml and 75mg/ml.

---

Lysozyme crystallization from Crystallization of Nucleic Acids and Proteins: A Practical Approach (1992) Edited by A. Ducruix and R. Giege, page 95.

Protocol 9. Testing crystallization using hanging drops.

1. Prepare stock solutions of3 M NaCl and 50 mg/ml (3.43 mM) lysozyme in 50 mM acetate at pH 4.5 and buffer stock solution (50 mM sodium acetate at pH 4.5). Filter all solutions with a microfilter of 0.22 microns.
2. Prepare a Limbro box as described in Section 4.2.1. [grease]
3. Fill up reservoirs [1 ml] of row A with solutions of NaCl ranging from 0.6-1.1 M by steps of 0.1 M.
4. On a coverslip, mix 4 [microliters] of protein stock solution with 4 microliters of reservoir. Flip it and set it on the greased rim.
5. Fill up reservoir [1 ml] of row B with solutions of NaCl ranging from 0.8-1.3 M by steps of 0.1 M. Repeat the experiment on row B after diluting protein of stock solution by a factor 2 to obtain a new one of 20 mg/ml (1.37 mM).
6. Fill up reservoir [1 ml] of row C with solutions of NaCl ranging from 1.0-2.0 by steps of 0.2 M. Repeat the experiment after diluting protein stock solution by a factor 2.
7. Use row D for duplicata or testing articular parameters (e.g. volume of drops to use the influence of kinetic effects on growth).
8. Store the experiments at 18° C.
9. Observe the experiments once a day for a week.

---

Lysozyme crystallization procedure

A. Protein solution preparation:

1. At room temperature, dissolve 20.0 mg of hen egg lysozyme in 0.25 ml of a 0.04 M pH 4.7 NaOac/HOAc buffer solution.
2. Dilute with 0.5 ml distilled water.
3. Add to this solution 0.25 ml of a 10% NaCl solution.

B. Well preparation:

1. Apply grease to the top of each well to be used for crystallization.
2. In the bottom of each well place 0.5 ml of a 0.04 M pH 4.7 NaOAc/HOAC buffer solution.
3. Add 0.5 ml of a 10% NaCl solution to each well.

C. Drop preparation:

1. Place 10 microliters of distilled water on a siliconized cover slide.
2. Drop onto the water drop 10 microliters of the protein solution above.

D. Crystallization set-up:

1. Invert the slide quickly.
2. Place the inverted slide, drop side down, over the well.
   **** Be careful not to contact the drop with the well sides****
3. Press the slide down onto the well until a complete seal is made between the slide and the well.
4. Place the crystallization plate in an undisturbed area.
5. Check the crystals daily--some should appear within 3 days.
6. Let sit for several days longer to allow crystals to anneal.

---

Crystallization of thaumatin

Chemicals Needed:

• Sigma (#T-7638) Thaumatin from Thaumatococcus Daniellii
• Sodium/Potassium Tartrate
• BisTris Propane buffer
• Ethylene Glycol

Procedure:

1. Prepare a 50 mg/ml sample of Thaumatin in distilled water.
2. Place 750 μL well solution (24% NaKTartrate (w/v), 15% ethylene glycol (v/v), and 0.1 M BisTris Propane pH 6.6) in each well of a Linbro or VDX tray.
3. On a clean silated cover slip, create a drop of 6μL Thaumatin solution and 4 μL well solution.
4. Seal this cover slip with grease over a well.
5. Crystals should grow overnight.

Variation:

Varying drop ratio, ethylene glycol concentration, or NaK Tartrate concentration should vary crystal size accordingly.

A cryoprotectant of 24% NaKTartrate (w/v), 25% ethylene glycol (v/v), and 0.1 M BisTris Propane pH 6.6 should be used to dip crystals for 20-60 seconds.

---

Crystallization of glucose isomerase

Chemicals Needed:

• Hampton Research (#HR7-102) Glucose Isomerase from Streptomyces rubiginosus
• Fluka Ammonium Sulfate ((NH₄)₂SO₄)
• HEPES buffer
• Distilled Water

Procedure:

1. Prepare a 20 mg/ml sample of Glucose Isomerase in distilled water (it will be necessary to use the centricon or dialysis to prepare the protein.
2. Place 750 μL of well solution (19% Ammonium Sulfate (w/v) in 0.1 M HEPES pH 7.2) in each well of a Linbro or VDX 24 well tray.
3. On a clean silated cover slip, create a drop of 3 μL Glucose Isomerase solution and 3 μL well solution.
4. Seal this cover slip with grease over a well.
5. Crystals may take a few days to grow, but should reach maximum size after approximately one week.

Variation:

Try growing the crystals in the presence of low concentrations (~ 5 mM) of Magnesium or Manganese compounds. Also try growing in the presence of various cryoprotectants (MPD seems to work well).

---

Trypsin crystallization procedure

Chemicals needed:

• Sigma (#T-8253) Bovine Pancreas Trypsin
• Amresco Benzamidine Hydrochloride
• Calcium Chloride
• Sodium Azide
• PEG4000
• Lithium Sulfate
• MES buffer
• Ethylene Glycol

Procedure:

1. Prepare 60 mg/ml sample of trypsin in 10 mg/ml benzamidine, 3 mM calcium chloride, and 0.02% (w/v) sodium azide.
2. Place 1 ml of well solution, 4% (w/v) PEG4000, 0.2 M Li₂SO₄, 0.1 M MES pH 6.5, and 15% ethylene glycol in each well of a Linbro or VDX tray.
3. On a clean silated cover slip create a drop of 3μ l protein solution and 3μ l of well solution.
4. Seal this cover slip with grease over a well.
5. Crystals should grow in 1 to 2 days.

Variations:

Use other drop ratios to increase or decrease crystal size.

Also try varying the concentration of the PEG4000 up to 8%.

---

Horse skeletal muscle myoglobin crystallization procedure

Chemicals needed:

• Calbiochem horse skeletal muscle Myoglobin #47592
• Ammonium Sulfate
• Sucrose
• Sodium Phosphate buffer
• Sodium Azide
• Distilled water
  

Procedure:

1. Prepare 20mg/ml sample of myoglobin in distilled water plus .02% (w/v) Sodium azide.
2. Linbro or VDX tray well solution consists of 1ml of 3.6M Ammonium sulfate and 50mM NaHPO₄ pH 5.3.
3. On a clean silated cover slip create a drop of 4μl myoglobin solution, 1μl 40% (w/v) sucrose, and 5μl of well solution.
4. Seal a cover slip with grease over each well.
5. Crystals should grow in about 3-5 days.

As far as a cryo-protectant we did a quick dip in 3.6M Ammonium sulfate, 50mM NaHPO₄ pH 5.3, and 15% (v/v) glycerol.

---

Ferritin

Chemicals Needed:

• Ammonium sulfate
• Tris Buffer
• Cadmium sulfate

Procedure:

1. prepare Ferritin
2. place 750 μl well solution ( 0.8M (NH₄)₂SO₄,0.1M Tris pH 7.5, 60mM CdSO₄) in each well of a Linbro or VDX tray
3. On a clean silated cover slip, create a drop of 4 μl protein and 2 μl well solution.
4. Seal cover slip with grease for each well
5. Crystals should grow in first 2 days but will continue to grow for 7-10 days

Variations:

To eliminate background precipitant mix protein and well in a micro centrifuge tube and spin down precipitant, apply a 6 μl drop of supernatant to cover slip. (These crystals tend to be smaller but cleaner).

To adjust size or number of nucleations vary CdSO₄ concentration or Ferritin concentration.

---

Proteinase K

Chemicals Needed:

• Sigma (#P-2308) Proteinase K from Tritrachium album
• Fluka Ammonium Sulfate ((NH₄)₂SO₄)
• Na Cacodylate buffer
• Tris Buffer
• Distilled Water

Procedure:

1. Prepare a 20 mg/ml sample of Proteinase K in a 25mM Tris buffer, pH 7.5
2. Place 500 μL well solution (1.2M (NH₄)₂SO₄ and 0.1 M Na Cacodylate pH 6.2) in each well of a Linbro or VDX tray.
3. On a clean silated cover slip, create a drop of 3 μL Proteinase K solution and 3 μL well solution.
4. Seal this cover slip with grease over a well.
5. Crystals should grow overnight.

Variation:

Other buffers can be substituted, i.e. HEPES works as well. Try varying the drop ratios to gain desired crystal size. Also PMSF can be added when making protein to lengthen shelf life of solution.