Practical tips for preventing proteins from "crashing out" & what you can do if it happens

Описание: Here are some tips to hopefully prevent your protein from crashing out on you (such as during dialysis). And, if it does happen, some things you can do.

Quick tips:
Avoid high concentrations
Don’t change things too drastically
Don’t go too low in your salt concentration
Troubleshoot on a small portion to find conditions that might make your protein happier

blog form: https://bit.ly/crashingout

First off - don’t freak out - sometimes things look way worse than they really are! A little bit of precipitate can precipitate a lot of worry (it might look super cloudy even though there is still a bunch dissolved)! But it should cause a bit of concern because it’s your proteins’ way of telling you it isn’t happy. We commonly call this “crashing out” - meaning your protein is coming out of solution (each protein molecule no longer has its own full coat of water, but rather clumps up with other protein molecules).

When this happens, it is commonly because you changed conditions on it too drastically - the big culprit is often salt (or more accurately, lack of it). It often happens during dialysis or some other form of buffer exchange. One of the most common purposes of dialysis (where you stick a protein in a membrane pouch it can’t escape from (but salts and other small things can) and stick that in a bath of a buffer you want to swap out then wait for diffusion to do its thing) is to remove salt. Such as removing excess salt before or after running ion chromatography, which relies on low concentrations of salt to allow your protein to bind to the little charged beads (resin) in the column, then high concentrations of salt to compete your protein off with salt-derived ions.

Often you do ion exchange after a “capture” affinity chromatography gel such as a nickel column that binds to a His- tag you put on your protein. When you do one of those columns you end up with a bunch of imidazole in your solution (the His mimic you use to compete your protein off). Even if you are using a different affinity strategy such as a Strep tag or GST tag, you typically use a fairly high salt concentration in order to prevent non-specific binding. (For reasons I get into here: http://bit.ly/ionicstrengthsalting, higher salt makes it harder for molecules to bind through charge-based attractions because the salt ions hide the charged parts from one another).

That high salt can get in the way of later things you want to do with the protein. So you often want to get rid of it. But not all of it. You don’t want it to be too low. And almost definitely don’t want it to be zero!

So instead of dialyzing or otherwise exchanging into no salt, just swap into low salt. But if you go too low, your protein will let you know - by precipitating. As the solution starts to get less solution-y, it gets cloudy.

Note: dialysis is just one method. Other ways of removing salt include desalting columns (which are low-res gel filtration (size-exclusion) columns, regular gel filtration, and buffer exchange using spin concentrators (ultrafiltration) - but this one is “dangerous” because it raises concentrations which can increase the risk of crashing out since it will be easier for protein molecules to find other protein molecules to bind to and harder for them to find water molecules).

Because it takes a while to remove things through dialysis (since it relies on passive diffusion - molecules just randomly moving around) you might not notice precipitation until you come in the next morning after setting up an overnight dialysis.

Often this happens after you left the lab all happy because you had a ton of protein. If that protein was squeezed into a small volume (i.e it was at high concentration) it makes it more prone to aggregation (more protein molecules to stick to, less free water to provide coats). So to try to avoid crashing out, avoid high concentrations and consider diluting your sample if you expect difficulty.

If you ran a metal affinity column, you might have some metal ions leached off into your solution. This can “help” his-tagged proteins precipitate since they can bind multiple his tags and therefore kinda bridge protein copies. To hide the metal from the protein, you can add a chelator like EDTA to your solution (as long as your protein isn’t metal-dependent, in which case this could make things worse!).

Now, what if things do crash out…

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