Photo courtesy of erin.
What is one of the most simplest things you can do for protein isolation to ensure a good prep?
Keep it cool, man.
Proteases, those sneaky, protein-nibbling enzymes, are more active at higher temperatures. Keeping your samples cool will thwart their amino-acid appetites.
Read on for some quick tips on keeping it cool.
If it comes in contact with your protein samples, get it cold first. Pre-label all tubes and keep them on ice (it’s harder to label cold/wet tubes), cool down the centrifuge prior to use, pre-chill any homogenizers, blades or instruments used for processing your samples, store all buffers in the refrigerator or put them on ice prior to protein isolation.
Now is not the time to take a minute to play Words With Friends. Work calmly, but expediently, to minimize the amount of handling of your samples. Have all your reagents and supplies at hand so you are not traipsing around the lab mid protein isolation.
Consider working in the cold
It’s not pleasant to spend the day in the cold room. But if you are working with large and/or very precious samples, you may want to hike up your thermal underwear, don a winter hat and spend a day getting acquainted with the long-forgotten items in cold storage.
Homogenize in multiple short bursts
Temperatures above 43°C will denature most proteins eventually while it only takes a few minutes at 95°C for complete denaturation. You might think your samples will never reach this temperature in your climate-controlled lab, but never underestimate the power of friction. Grinding, sonicating, beating – mechanical methods for physically disrupting tissues and cells warms up samples and can cause localized heat spots. Reduce the heat by performing multiple short rounds instead of a long continuous one. And don’t forget to put your samples on ice in between rounds.
Freeze your samples quickly prior to storage. Use a dry ice/ethanol bath or freeze in liquid nitrogen.
Store in small aliquots
Multiple freeze/thaw cycles will quickly denature and destabilize proteins. Store samples in multiple small aliquots to limit the number of times the sample is frozen and thawed.
Keep them in the deep freeze
For long-term storage, keep your protein samples at-80°C or in liquid nitrogen.
If all else fails, just chill out.
Photo courtesy of Ian Burt.