Do you need your protein in its native form, intact, with full functionality? Do you need to isolate organelles and nuclear fractions from the cytoplasm? Or do you need a slurry of everything in your cell or tissue? Whatever your experiment, you can maximize the amount of functional, detectable or active proteins by handling your proteins with care!
Here are the correct ways to handle you protein sample, no matter the reason:
Keep it CLEAN!
Most likely your proteins are coming from cell extracts, either from cell culture or harvested tissue. Either way, you’ll likely use a protease such as trypsin to dissociate the cells. And what do proteases do? Degrade proteins – exactly what you do NOT want to happen to your-favorite-protein. So make sure you wash off the trypsin (or other protease) from your dissociated cells before lysing them.
Keep it GENTLE!
Cell lysis is a violent affair. You can lyse through mechanical and/or chemical means. Mechanical lysing is excellent when you have a large cell pellet or piece of tissue, or if you cannot use harsher lysis buffers. Some methods of mechanical lysis include using a mortar and pistil, pipetting using a very narrow pipette, or sonication. Chemical
lysis depends on lysis buffers to break open the cell wall and/or membrane to release all the contents of the cell. It does not involve grinding or scraping, but rather uses detergents, pH, salts, and enzymes to lyse cells.
How you choose to lyse your cells depends on your downstream experiment. Do you plant on performing a native gel to detect intact protein:protein bonds, looking to fractionate the organelles, or optimizing enzymatic assays? You’ll want to be gentle as can be. Doing a Western Blot? You can be a little more aggressive, but still be careful. Most labs and suppliers of protein assay materials have helpful suggestions for which lysis method works best for your experiment. For more about cell lysis see Emily Crow’s article “
How to Lyse Cells for Protein Extraction.”
Keep it COLD!
As soon as you lyse that first cell, the clock will be ticking on your protein’s lifespan. A simple way to slow down your protein’s denaturation or degradation is to keep it cold. As soon as intact cells are disrupted, all your solutions should be cold. Buffers, PBS, detergents, everything should be kept cold, and minimize the time your protein spends off the ice.
Keep it BUFF!
As soon as cells are lysed, all the gooey insides of cells are now mixing and churning together. Therefore, the buffers you choose are critical here to minimize the damage endogenous proteases, improper pH, and other sudden changes caused by this mixing can do to your protein. The buffer you choose will depend on what type of cells you’re using: mammalian, yeast, insect, plant, bacteria. If you use mechanical methods to lyse your cells, you might use a gentler buffer. If you don’t care if your protein is denatured, but want it whole, you can use a buffer with stronger detergents. Many lab-supply companies have pre-made buffers and guides that help take some of the guess work out of which buffer is right for your cells and experiment. For more about protein purification buffers see Jennifer Cable’s article “
How to Design the Perfect Protein Purification Buffer.”
Keep it FRESH!
Use fresh protease inhibitors every time. It might sound fussy, but most protease inhibitors are enzymes and they loose efficacy over time. Most
protease inhibitors come in concentrated form, so diluting a fresh aliquot into your lysis buffer just before you’re ready will ensure your favorite protein will have full protection.
From protein immunoblots to ELISA to functional assays, the most important thing in protein biochemistry is high-quality proteins. If you handle your tissue and cell extracts with care, you’re sure to get great results with your downstream application. Do you have any tips of your own? Comment below.
