Hampton research HR2-112 Crystal Screen 2 说明书

User guide

Crystal Screen 2™ is a complete reagent kit designed to provide a rapid screening method for the crystallization of biological macromolecules. Crystal Screen 2 is a straightforward, effective, and practical kit for deter- mining preliminary crystallization conditions. Crystal Screen 2 is also ef- fective in determining the solubility of a macromolecule in a wide range of precipitants and pH.

Crystal Screen 2 is a sparse matrix an extension of trial crystallization re- agent conditions based upon the original Jancarik and Kim screen (3). The primary screen variables are salt, pH, and precipitant (salts, polymers, vola- tile organics, and non-volatile organics).

Sample Preparation

The macromolecular sample should be homogenous, as pure as is prac- tically possible (>95%) and free of amorphous and particulate material. Remove amorphous material by centrifugation or micro-filtration prior to use (1, 2, 4).

The recommended sample concentration is 5 to 25 mg/ml in water. Ini- tially, the sample should be free of any unnecessary additives in order to observe the effect of the Crystal Screen 2 variables. Ideally, the initial screen should be performed with a sample which has been dialyzed against water although ligands, ions, reducing agents, or other additives may be present as required by the sample for solubility, stability, or activity.

Screen 2 reagent 2 into reservoir A2. Repeat the procedure for the remaining 46 Crystal Screen 2 reagents using a clean pipet tip for each reagent so as to avoid reagent contamination and carry over.

3. Pipet2 µl of the sample to the center of a clean, siliconized 22 mm diam- eter circle or square cover  See Figure 2.

Since it is the most frequently reported method of crystallization, the fol- lowing procedure describes the use of Crystal Screen 2 with the Hanging Drop Vapor Diffusion method. Crystal Screen 2 is also very compatible with the Sitting Drop, Sandwich Drop, Microbatch, and Microdialysis methods. A complete description of the Hanging, Sitting, Sandwich Drop, Dialysis and other crystallization methods are available from the Hampton Research Crystal Growth 101 Library.

1. Prepare a VDX Plate (HR3-140) for Hanging Drop Vapor Diffusion by ap-plying a thin bead of cover slide sealant to the upper edge of each of the 24  One may also use a Greased VDX Plate (HR3-170). Forty-eight reservoirs are to be prepared for a complete Crystal Screen 2. See Figure 1.

Technical Support
Inquiries regarding Crystal Screen 2 reagent formulation, interpretation of screen results, optimization strategies and general inquiries regarding crystallization are welcome. Please e-mail, fax, or telephone your request to Hampton Research. Fax and e-mail Technical Support are available 24 hours a day. Telephone technical support is available 8:00 a.m. to 4:30 p.m. USA Pacific Standard Time.

How to Reproduce Crystal Screen 2 Reagents
Crystal Screen 2 reagents and optimization conditions based on Crystal Screen 2 hits can be formulated using volumetric methods and carefully prepared reagent stocks (Table 1). Note the examples below.
Example 1. To prepare 1.0 milliliter of Crystal Screen 2 reagent 1 in a crystallization plate.Solution Composition: 10 % w/v Polyethylene glycol 6,000 2.0 M Sodium chloride
• 400 ml water 3
• 200 ml 50 % w/v Polyethylene glycol 6,000
(CAS # 25322-68-3, Catalog # HR2-533)
• 400 ml 5.0 M Sodium chloride
(CAS # 7647-14-5, Catalog # HR2-637)
Make no pH adjustments. Mix well by aspirating and dispensing the solution multiple times.
Example 2. To prepare 10 milliliters of Crystal Screen 2 reagent 24.Solution Composition: 30% v/v Jeffamine ® M-600 ®
0.1 M MES monohydrate pH 6.5
0.05 M Cesium chloride
• 2.5 ml water 3
• 0.5 ml 1.0 M Cesium chloride
(CAS # 7647-17-8, Catalog # HR2-719)
• 1.0 ml 1.0 M MES monohydrate pH 6.5
(CAS # 145224-94-8, Catalog # HR2-787)
• 6.0 ml 50% v/v Jeffamine ® M-600 ® pH 7.0
(CAS # 77110-54-4, Catalog # HR2-501)
Make no pH adjustments. Mix well.3 ASTM Type II (laboratory grade) or Type III (analytical grade) water.
Formulation Notes for Crystal Screen 2 Reagents
1. No additional pH adjustment is made to any reagent after formulation.
Use the buffers in Table 1 to reproduce a Crystal Screen 2 reagent.
2. All Optimize solutions and screen reagents are sterile filtered using 0.22
um filters into sterile containers.
3. Add water first as this will help maintain the solubility of subsequently
added reagents.

4. When formulating reagents using a pipet, add the largest volume
last (except water). Use this larger volume setting to aspirate and
dispense the reagent until the solution is mixed.
5. When formulating reagents using a pipet, use a clean, sterile pipet tip
for each reagent added to the solution.
6. Use the buffers in Table 2 to systematically vary the pH as a crystalliza-
tion variable.
pH as a Crystallization Variable
The buffers listed in Table 2, can be used to vary the pH as a crystallization variable and are recommended when optimizing a crystal grown from a Crystal Screen 2 kit.
Optimize™ buffer stocks are supplied as a 100 milliliters sterile filtered solution. The pH can be adjusted to the indicated pH range using either HCl or NaOH and the supplied titration tables.
StockOptions™ buffer kits contain 10 milliliters each of ready to pipet buffers, titrated in 0.1 pH increments over the indicated pH range. The number of reagents offered in a StockOptions buffer kit depends upon the pH range of the buffer. The broader the pH range, the more buffers in the kit.
Online Information
Visit www.hamptonresearch.com and enter one of the following:
• Reagent Catalog Number
• Kit Catalog Number
• CAS Number
• Reagent Name
To obtain reagent specifications, pH titration tables, user guides, certificates of analysis, material safety data sheets (MSDS), and any other additional information.
MakeTray™
MakeTray is a free, web based program at www.hamptonresearch.com which generates both a pipetting worksheet and a reagent formulation document for crystallization set ups. MakeTray allows one to enter general information about the sample and experiment, which is then printed on the pipet worksheet and the reagent formulation document. The plate size can be customized for any number of wells, so MakeTray works for: 24, 48, and 96 well plates. MakeTray is especially useful for the design and formulation of crystal optimization experiments.
TM
Crystal Screen 2 Fundamentals HR2-112 (pg 2)
Solutions for Crystal Growth
Each of these reagents are available as an Optimize™ crystallization grade reagent from Hampton Research. Table 1 provides the common chemical name, the Hampton Research catalog number, supplied stock concentration, the supplied volume, and the CAS number for each reagent. For more information on a specific Optimize reagent,

Individual StockOptions buffers titrated to any pH within the kit’s pH

range are available in 185 ml volumes from the Hampton Research Custom Shop