The centrifuge is one of the essential pieces of equipment for protein expression.

Release time:

2020-12-17

Overview:

To successfully complete protein expression, you can’t do without a centrifuge. As the saying goes, “Details determine success or failure”—especially in complex experiments like protein expression and purification. Even a minor operational mistake can directly affect the final outcome.

　　You can't get through protein expression without it. Centrifuge As the saying goes, “Details determine success or failure,” especially in complex experiments like protein expression and purification. Even a minor operational mistake can directly affect the final outcome.

　　Although it can be roughly divided into three steps—expression, separation, and purification—the process is quite cumbersome.

　　Before protein expression, there’s still a series of complex processes that take a long time. Even at the final step, we can’t let our guard down.

　　Protein purification is the final step in the purification process. Selecting an appropriate purification method can enhance the efficiency of protein purification, making the process simple and convenient. Commonly used methods include affinity chromatography, ion-exchange chromatography, gel-filtration chromatography, and hydrophobic interaction chromatography.

　　Affinity chromatography

　　Affinity chromatography purification is a technique for protein purification that exploits the property of biological macromolecules to reversibly bind with high specificity to certain corresponding molecules.

　　Ion-exchange chromatography

　　Ion-exchange chromatography is a technique used to separate and purify proteins based on the differences in the amount of charge carried by their surfaces.

　　Gel filtration chromatography

　　Gel filtration (also known as size-exclusion chromatography or molecular sieving) is a method for separating proteins from a mixture based on their molecular size.

　　Hydrophobic Interaction Chromatography

　　Hydrophobic interaction chromatography is a chromatographic method that separates sample molecules based on the differences in hydrophobic forces between the hydrophobic groups of the sample molecules and the hydrophobic ligands of the chromatographic medium in a salt-water system.

　　Although there are many different methods, all of them require the use of a centrifuge for separation and purification.

　　Take the induction of recombinant lipoxygenase protein as an example:

　　1. Inoculate the E. coli BL21 strain containing recombinant lipoxygenase protein into 5 mL of LB liquid medium (containing 100 μg/mL ampicillin), and incubate with shaking at 37℃ overnight.

　　2. Dilute the overnight culture at a ratio of 1:50 or 1:100. Typically, transfer 1 mL of the overnight culture into 100 mL of LB liquid medium containing 100 μg/mL ampicillin, and shake at 37℃ until the OD600 reaches 0.6–0.8 (preferably 0.6; this usually takes about 3 hours). Take a 10-μL sample for SDS-PAGE analysis.

　　3. The control group was not treated with any inducer, while the experimental group was supplemented with IPTG to a final concentration of 0.5 mmol/L and further incubated at 37℃ for 1–3 hours.

　　4. Centrifuge at 12,000 rpm for 10 min; discard the supernatant and store the cell pellet in a freezer at -20℃ or -70℃.

　　The above is a comprehensive explanation of protein expression. The main point this article aims to convey is that in any experiment, details are crucial to its success or failure. Although human factors can be deliberately controlled, attention to the details of auxiliary equipment remains essential—choosing the right centrifuge is particularly critical.

Blood separation and purification rely on a low-speed centrifuge.

Washing red blood cells with a low-speed centrifuge is convenient and quick.