New Graduate Lab Survival Guide! Extraction of proteins from cells and tissues
Originally published as The New Graduate Lab Survival hemp extraction centrifuge 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 bra tape measure 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 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 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 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) cbd centrifugal extractor 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 15ml centrifuge tube with a pipette place it into a centrifuge at 4 degrees (precool the centrifuge in advance) centrifuge for 15min 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
