Protein crystallization screening 96 condition kit using polyethylene glycol as precipitator

A technology of polyethylene glycol and kits, which is applied in the direction of enzyme and peptide preparation methods, biochemical equipment and methods, etc., and can solve problems such as low success rate

Inactive Publication Date: 2010-09-01
海安华达石油仪器有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In order to overcome the deficiency that the existing 96-condition screening kit has a low success rate in protein crystallization condition screening, the present invention provides a 96-condition kit using polyethylene glycol as a precipitating agent for protein crystallization screening, which can improve protein crystallization conditions Screening success rate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Concanavalin A was screened using the PEG Grid Screen 96 prepared above.

[0031] Step 1: Dissolve concanavalin with product number L7647 from Sigma, USA in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid- In Na buffer solution, prepare a concanavalin solution with a concentration of 20 mg / ml, and filter out impurities with a 0.22 μm filter membrane.

[0032] The second step: use the above 96 conditions to screen concanavalin crystallization conditions.

[0033] Step 3: Use a sitting drop plate, add 90 μl of pool solution to each deep well, use an automatic pipetting system to mix 1 μl of screening crystallization agent and 1 μl of protein solution into a crystallization drop, drop it into the small sitting drop hole, and seal the crystallization plate .

[0034] Step 4: After the above-mentioned crystallization solution system was placed in a temperature-controlled box at 20° C. for 7 days, the number of conditions under which concanavalin crystals app...

Embodiment 2

[0036] Example 2: Condition screening of lysozyme (lysozyme) was carried out using the PEG Grid Screen96 prepared above.

[0037] Step 1: Dissolve the lysozyme with product number 100940 from Japan Seikagaku Company which has undergone six recrystallizations in N-2-hydroxyethylpiperazine-N'-2- Prepare a lysozyme solution with a concentration of 40 mg / ml in ethanesulfonic acid-Na buffer solution, and filter out impurities with a 0.22 μm filter membrane.

[0038] The second step: use the above-mentioned 96 conditions to screen the crystallization conditions of lysozyme.

[0039] Step 3: Use a sitting drop plate, add 90 μl of pool solution to each deep well, use an automatic pipetting system to mix 1 μl of screening crystallization agent and 1 μl of protein solution into a crystallization drop, drop it into the small sitting drop hole, and seal the crystallization plate .

[0040] Step 4: After the above-mentioned crystallization solution system was placed in a temperature-cont...

Embodiment 3

[0042] Example 3: Conditional screening of proteinase K (proteinase K) was carried out using the PEG Grid Screen96 prepared above.

[0043] Step 1: Dissolve proteinase K with product number P6556 from Sigma, USA in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Na buffer with a pH of 7.0 and a concentration of 0.025mol / L In the solution, a proteinase K solution with a concentration of 30 mg / ml was prepared, and impurities were filtered out with a 0.22 μm filter membrane.

[0044] Step 2: Use the above 96 conditions to screen proteinase K crystallization conditions.

[0045] Step 3: Use a sitting drop plate, add 90 μl of pool solution to each deep well, use an automatic pipetting system to mix 1 μl of screening crystallization agent and 1 μl of protein solution into a crystallization drop, drop it into the small sitting drop hole, and seal the crystallization plate .

[0046] Step 4: After the above-mentioned crystallization solution system was placed in a temperature-co...

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Abstract

The invention discloses a protein crystallization screening 96 condition kit using polyethylene glycol as a precipitator and aims to solve the technical problem existing in the conventional 96 condition screening kit that the success rate of protein crystallization condition screening is low. The PEG adopted in the technical scheme is the top eight types PEG which are most frequently used according to the statistical data of a BMCD database rather than selected randomly, so that the crystallization success rate of biological macromolecules is obviously improved. For example, the success rate of canavaline is increased from 4.2 percent in background technology to 81.2 percent, the success rate of lysozyme is increased from 29.2 percent in background technology to 46.9 percent, the success rate of protease K is increased from 58.3 percent in background technology to 66.7 percent, and the success rate of catalase is increased from 15.6 percent in background technology to 30.2 percent. In addition, the kit uses the PEG as the precipitator, a few types of reagents are used, and compared with the background technology kit, the kit of the invention is lower in cost.

Description

technical field [0001] The invention relates to a 96-condition kit for protein crystallization screening, in particular to a 96-condition kit for protein crystallization screening using polyethylene glycol (PEG) with different molecular weights as precipitation agents. Background technique [0002] Hampton Research's public name is Index TM The specific composition of the 96-condition screening kit is as follows, including three parts, precipitant, salt, and buffer. The precipitant mainly includes ammonium salt, sodium salt, magnesium salt, phosphate, formate, organic matter, polymer, etc., and its concentration is relatively high, about 2M; the salt mainly includes sodium salt, magnesium salt, calcium salt, sulfate , acetate, tartrate, etc., the concentration is low, below 1M; the buffer solution includes 6 kinds, the concentration is 0.1M, and the pH value ranges from 3 to 9. [0003] Table 1: Index TM 96 condition ingredient list [0004] serial number ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C12N9/00
Inventor 尹大川张仙芳郭云珠鹿芹芹刘君
Owner 海安华达石油仪器有限公司
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