6xHis Labeled Protein Nickel Column Purification Method Protocol
Release time: 2016-08-30
1. Solution preparation:
1.1 Binding Buffer: 10mM PB, 500mM NaCl, 10mM imidazole, pH7.4.
1.2 Elution Buffer1: 10mM PB, 500mM NaCl, 50mM imidazole, pH 7.4.
1.3 Elution buffer 2 (Elution Buffer2): 10 mM PB, 500 mM NaCl, 250 mM imidazole, pH 7.4
1.4 Elution buffer 3 (Elution Buffer3): 10 mM PB, 500 mM NaCl, 500 mM imidazole, pH 7.4
1.5 Denaturing buffer solution: 10mM PB,500mM NaCl,8M urea, pH7.4.
1.6 CIP solution: 0.5M NaOH,1M NaCl
1.7 Chromatographic packing: Ni Crystarose FF
2. Sample preparation:
2.1 Prokaryotic intracellular soluble expression samples:
a) After induction of expression, the bacteria were centrifuged at 7000 rpm for 30 min, and the supernatant was discarded.
b) Resuspend the bacterium with Binding Buffer.
c) Lysis by ultrasonic crusher for 30 min.
d) Centrifuge at 10,000rpm for 15min and remove the supernatant.
3、 6xHis tag protein affinity chromatography
3.1 Equilibrate the chromatographic column with 5 times the volume of Binding Buffer.
3.2 Apply the sample at a flow rate of 150cm/h.
3.3 Wash the column with 3--5 times the volume of Binding Buffer until the column is parallel to the baseline at a flow rate of 300 cm/h.
3.4 Wash the column with 3-5 volumes of Elution Buffer1, at which time all impurities such as heteroproteins and nucleic acids are eluted, at a flow rate of 300 cm/h.
3.4 Phase elution, elute with Elution Buffer2 and Elution Buffer3 respectively, and collect the eluate.
4、 CIP
4.1 Rinse the chromatographic column with 5 - 10 volumes of deionized water at a flow rate of 600 cm/h.
4.2 Rinse the chromatographic column with 5 times the volume of CIP solution at a flow rate of 75 cm/h. The flow rate should not be too fast at this time to prevent damage to the column.
4.3 Flush the column with deionized water at a low flow rate of 75 cm/h for 5 volumes, and then flush the column with deionized water at a high flow rate of 600 cm/h for 5 volumes.
4.4 Rinse the column with 20% ethanol solution for 5 volumes at a flow rate of 300 cm/h.
4.5 Storage.
Recommended news
Keep abreast of company news and industry news
In January 2017, the Development and Reform Commission issued the "13th Five-Year Plan" for the development of the biological industry, which pointed out the direction of the key issues and
6xHis Labeled Protein Nickel Column Purification Method Protocol
1. Solution preparation: 1.1 Binding Buffer: 10mM PB, 500mM NaCl, 10mM imidazole, pH7.4. 1.2 Elution Buffer1: 10mM PB, 500mM NaCl, 50mM imidazole, pH 7.4.
Jingcheng CrystaroseC FF filler test report
1、Apparatus and consumables 1.1 Instrument: JC-1 automated protein purification system (Jingcheng) 1.2 Chromatographic column: XK16 (GE) 1.3 Jingcheng Biotechnology: CrystaroseC FF(5ml)
Metal chelate chromatography affinity packing comparable to imported products
CrystaroseC FF metal chelate chromatography affinity packing has super high rigidity, higher linear flow rate up to 700cm/h, and the back pressure is close to the imported similar products or even lower. Therefore, the packing life is longer.
Contact us
E-mail：122460871@qq.com
Address: Building B, R&D Area, Wuhan National Biological Industry Base Project, No. 666 Gaoxin Avenue, East Lake High-tech Development Zone, Wuhan City
Copyright © Wuhan Jingcheng Biotechnology Co., LTD. Business License SEO This site supports IPV6