The purified pro-tHAP sample was concentrated to 10 mg-mL−1 and used for crystallization. Crystallization screens were set up with a Phoenix crystallization robot (Protein Crystallography Facility, IIT Bombay) using commercial screens (JCSG CORE I and PEG-Suite (Qiagen, Hilden, Germany); INDEX, PEG-Ion and PEG-Rx (Hampton Research)), by the sitting drop vapor diffusion method at 295 K. Each well contained 0.3 μL protein and 0.3 μL of reservoir solution, equilibrated against 50 μL of reservoir solution. Crystals of pro-tHAP were obtained in three different crystallization conditions: (a) 200 mM ammonium citrate tribasic pH 7.0, 100 mM imidazole pH 7.0, 20% w/v polyethylene glycol monomethyl ether 2000; (b) 200 mM lithium citrate tribasic tetrahydrate, 20% w/v polyethylene glycol 3350; and (c) 200 mM ammonium phosphate dibasic, 20% w/v polyethylene glycol 3350. The crystals grew to their maximum size within 4–5 days. The pro-tHAP structures solved with the crystals obtained in crystallization conditions a, b, and c are referred to here as HAP-zymo1, HAP-zymo2, and HAP-zymo3, respectively.
Jcsg core 1
Manufactured by Qiagen
Sourced in Germany
The JCSG CORE I is a laboratory equipment product designed for protein crystallization. It provides a standardized set of chemical conditions to facilitate the initial screening of protein samples for potential crystallization. The JCSG CORE I kit contains a variety of buffers, salts, and precipitants that can be used to systematically explore different crystallization conditions.
Automatically generated - may contain errors
2 protocols using jcsg core 1
1
Pro-tHAP Crystallization Conditions
2
Crystallization of S-GAT Fab Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The complex crystal of S-GAT Fab and corresponding drug was prepared by co-crystallization method with a molar ratio of 1 : 10. All the crystals were generated by the vapor diffusion method in sitting drops at 20 °C. Initial crystal screening was performed with 8 kits: JCSG core I, JCSG core II, JCSG core III, JCSG core IV, JCSG classic, JCSG +, PEGs I and PEGs II (QIAGEN, Hilden, Germany). The protein drops for initial crystal screening contained 0.1 μL Fab fragment and 0.1 μL reservoir solution were dispersed on 96-weill plates using Mosquito (TTP LabTech, Royston, U.K.). The crystals obtained by screening were determined as protein crystals by using Izit Crystal Dye (Hampton Research, Aliso Viejo, CA, U.S.A.), and then the corresponding conditions were optimized by adjusting the buffer pH and the precipitant concentration. The protein drops for optimization contained 1 μL Fab fragment and 1 μL reservoir solution, and crystals appeared after 30 d. The optimized reservoir condition of the S-GAT Fab apo form crystal contained 1.6 M ammonium sulfate, 24% glycerol and 0.8 M sodium acetate, pH 4.6. The optimized reservoir condition of S-GAT Fab crystal in complex with S-GAT (S-GAT Fab complex) contained 0.2 M sodium chloride, 1.4 M ammonium sulfate and 1 M sodium acetate, pH 4.9.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!