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Affinity chromatography of centrifuges

Sep 03, 2020

Affinity chromatography (AFFINITY chromatography) is a technique for protein purification by using biological macromolecules with reversible binding properties of some corresponding molecules.


Ion exchange chromatography


Ion exchange chromatography (ION-exchange chromatography) is a technique for protein separation and purification according to the charge on the surface of the protein.


Gel filtration chromatography


Gel filtration (also known as exclusion chromatography or molecular sieves) is a method of separating proteins from a mixture based on the size of the molecules.


Hydrophobic chromatography


Hydrophobic chromatography is a kind of chromatography method which uses the hydrophobic differences between the hydrophobic groups of the sample molecules in the salt-water system and the hydrophobic ligands of the chromatography medium to separate the samples.


Although there are various methods, all of them need to be separated and purified by a centrifuge.


Take the induction of lipoxygenase recombinant protein as an example:


1. Inoculate Escherichia coli BL21 strain containing recombinant lipoxygenase protein in 5 mL LB liquid medium (containing 100 ug/mL ampicillin) and culture overnight at 37℃ by shock.


2. Overnight bacteria were diluted at 1:50 or 1:100, and generally transferred to 1 mL overnight culture medium in LB liquid medium containing 100 mL (including 100 ug/mL ampicillin), and incubated at 37℃ till OD600 = 0.6-0.8 (preferably 0.6, about 3 h).

10 ul samples were taken for SDS-PAGE analysis.


3. No inducer was added to the control group, and IPTG was added to the experimental group until the final concentration was 0.5mmol/L, and the culture was continued at 37℃ for 1-3h.


4. The centrifuge was centrifuged at 12000 RPM for 10 min, and the supernatant was discarded. The precipitation of the bacteria was stored in the refrigerator at -20℃ or -70℃.


The above is the explanation of protein expression. The focus of this paper is to express that the details of the success or failure of an experiment are crucial. Although the manual aspect can be controlled manually, more attention should be paid to the details of auxiliary equipment, and choosing a suitable centrifuge is crucial.


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