New Graduate Lab Survival Guide! Extraction of proteins from cells and tissues

Commentaires · 83 Vues

New Graduate Lab Survival Guide! Extraction of proteins from cells and tissues

Originally published as The New Graduate Lab Survival Guide! Extraction of proteins from cells and tissues It's the beginning of the school season again, and the students are all going to school, so they can study happily again. Expand the full text Today, I will introduce some introductory experimental knowledge to you: In the course of the experiment, we often need to prove whether some proteins in the experimental group are more or less than those in the control group, so we need to extract proteins from cells or tissues for measurement. Today, we will introduce the basic knowledge and steps of protein extraction from cells and tissues. Principles of protein sample preparation: 1. Keep it as simple as possible To avoid protein loss; 2. Cell and tissue samples should be prepared to minimize protein degradation Low temperature and protease inhibitors can prevent the degradation of protein; 3. Sample lysate should be freshly prepared , subpackage and store at -80 ℃, do not freeze and thaw the prepared sample repeatedly; 4. Remove remaining impurities by ultracentrifugation 。 General understanding methods are divided into mild lysis methods and violent protein lysis methods. Mild cracking method It is used to make up a relatively simple sample or to analyze a specific organelle. (1) Osmotic lysis: blood cells, tissue culture cells (2) Freeze-thaw cracking: bacteria and tissue culture cells; liquid nitrogen freeze-thaw (3) Detergent lysis: use detergent to lyse cell membranes, lyse cells, and release contents; used to organize cell (4) Enzymatic lysis of cells: cells containing cell walls such as plants, bacteria,wiped film distillation, and fungi. Violent proteolytic method It is often used for cells that are difficult to break, such as cells in solid tissues, or cells with rigid cell walls, such as yeast cells. (1) ultrasonic lysis method: cell sample (2) Freund's press: a shear force produced by forcing cells through a small aperture under high pressure, thereby lysing the cells; often used for microorganisms,jacketed glass reactor, algae, (3) Grinding method: usually used for solid tissues and microorganisms; frozen in liquid nitrogen and then ground into powder. (4) mechanical homogenization method: (5) Glass bead homogenization: breaks cell walls by vigorously shaking glass beads; used for cell suspensions or microorganisms. Then the steps of protein extraction come: Required equipment: low temperature high speed centrifuge, homogenizer, ultrasonic instrument, vacuum pump, cell hanging spoon, etc. Required reagents: RIPA lysate, protease inhibitor (PMSF), sterile PBS, cbd centrifugal extractor ,rotovap distillation, etc. Protein sample extraction 1 Extraction of cellular proteins: 1. Take out the cell culture plate to be extracted according to the needs of the experiment. ? Place on ice and remove the supernatant with a suction device. PS: All operations should be carried out on ice. 2. Cell per pore (Six-well plate) 1-2 ml of precooled PBS solution at 4 ° C was added. Lay flat and gently shake for ten seconds to wash the cells, then aspirate the wash solution, washing the cells three times (in order to remove the remaining supernatant), and do not aspirate the supernatant in the last time. 3. Scrape the cells gently with a spatula. Then, transfer the cells and supernatant into a 1.5ml centrifuge tube with a pipette, place it into a centrifuge at 4 degrees (precool the centrifuge in advance), centrifuge for 1.5min, and discard the supernatant. Aspiration and transfer of cells and supernatant: The hanging spoon looks like this: 4. Lysate was prepared (The lysate used is RIPA lysate, which can fully release the protein.) Add 10 μl of PMSF (100 mM) per 1 ml of lysate, shake well and place on ice. (PMSF should be shaken until there is no crystal before mixing with the lysate). PS: PMSF is a protease inhibitor, which can reduce protein release. Other protease inhibitors can also be cocktail, NaF, NaN3, etc., and multiple protease inhibitors can also be added at the same time. 5. Adding the prepared lysis solution into the first and the second lysis solution, In the cell pellet obtained in step 3, 510 ^ 6 cells are generally added to 100ul of lysis solution, and then blown and resuspended to fully lyse the cells. 6. The cells were further lysed with a sonicator The cells were placed on ice for ultrasound. Generally, the power was selected to be about 15%, and the lysis time was 1 minute (ultrasound for 2S, rest for 4s). When the centrifuge tube was still turbid after one ultrasound, the cells could be sonicated again at an interval of several minutes. 7. After the ultrasound The cells were centrifuged at 12000 rpm for 25 min in a 4 degree centrifuge. 8. The centrifuged supernatant was sub-packaged and transferred to 1.5ml centrifuge tubes were stored at -80 ℃ for later use. 2 Extraction of total protein from tissue: 1. Place a small amount of tissue mass in Add 500ul of strong RIPA lysate containing PMSF into a 1.5ml centrifuge tube, first use clean scissors to cut the tissue block as much as possible, when the tissue is soft (such as brain tissue), then use a 1ml gun head to continuously blow and suck, then use a homogenizer to further break the tissue, and finally use an ultrasonic instrument to carry out ultrasonic lysis; When the tissue is tough (such as muscle tissue), the homogenizer can be directly used to break the tissue, and finally the ultrasonic instrument can be used to carry out ultrasonic lysis. (Dissecting tissue is to make tissue pieces smaller and smaller by different methods.). 2. Same method as above ? Put the centrifuge tube into a 4 degree centrifuge, centrifuge at 12000 rpm for 25 min,50l rotovap, and take the supernatant into a 1.5ml centrifuge tube and store it at -80 ℃. Ok, that's all for today. To be continued.. "Medical School" has always been committed to serving "medical scholars", pushing the most cutting-edge and valuable original articles on clinical and scientific research to clinicians and researchers. Return to Sohu to see more. Responsible Editor:. toptiontech.com

Commentaires