Want to go on a field trip to a PROTEIN CHROMATOGRAPHY museum? I often think about the proteins I express as my “kids” and when I go to pick them up from the cells I express them in, they’re hanging out with a bunch of friends (contaminating proteins) I don’t want to take home. So I send all of them on field trips through a series of museums until we’re finally united.

note: originally posted September 2, 2019; refreshed & video added 3/19/22

if you want a more serious explanation: http://bit.ly/proteincleaning

PROTEIN CHROMATOGRAPHY is a method used to purify proteins by taking advantage of their different properties by sending them traveling through a CHROMATOGRAPHY COLUMN – which is just a tube filled with little beads (RESIN). 

I like to think of it kinda like sending people through a museum that takes them different amounts of time to go through and catching them as they come out. They go in the entrance (top of the column) and out the exit (elute from the bottom of the column) where you can use a UV monitor to see where protein comes out (like tracking when people exit).

Proteins absorb at 280nm so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. And the computer shows this to us as a peak. more here: https://bit.ly/chromatograms

But *all proteins* do this, not just the one you’re interested in. So it can tell you how many people are coming out at different times, but not whether they’re Bobs or Joes or a mixture. As they come out we use a fractionator to divert them into different wells in a deep-well plate based on when they come out (like putting everyone who comes out of the museum between 4 & 4:15 in one bus, 4:15 to 4:30 in another, etc.)

Then we use SDS-PAGE to “unwind” & separate the proteins in the different fractions by their length (in the chromatography steps we want to avoid that unwinding (denaturation) but now it’s important because wound proteins may be bound to one another and/or their shape may make them not act their true size. This can show us what size the proteins under the peak are. And if you want to really make sure that something is really your protein not something of similar size you can run a Western Blot where you use antibodies specific to your protein to probe for it after transferring it to a probe-able membrane.

So, what determines when the protein comes out? 

There’s no going backwards because you’re pumping liquid through (if you’re using a liquid handling machine like this AKTA with pre-packed columns) or gravity’s pushing it down if you’re using self-packed gravity flow columns. More about how the AKTA works here: https://bit.ly/aktainaction

Speaking of which, when they’ll come out the exit depends on how fast you’re pumping liquid (flow rate), how long a path they take through the museum which depends on how many “extra exhibits” they go through (this is the approach size exclusion chromatography (SEC), aka gel filtration, takes), and how much they like the placards on the museum’s walls (the AFFINITY CHROMATOGRAPHY approach)

As my family can attest to, I can spend hours in a science museum – you basically have to force me out. So if they were to wait at the exit they’d be waiting forever. So I think they’ve come up with a clever scheme whereby they convince a school-full of loud, pushy kids to enter and compete with me for reading the placards. 

But we all have different tastes and, while I love science museums, I couldn’t wait to get out of some museums my family has wanted to go to. So we choose what museum to send the proteins through based on their interests. The more specific to the protein *we’re* interested in, the better. A general science museum will attract a lot of people, whereas a museum devoted to my favorite topics would just (sadly) attract me. 

Proteins’ “interests” depend on the properties of the amino acid letters they’re made up of. Some of these letters can be charged (depending on the pH), so each protein has an overall charge at any given pH. more here: https://bit.ly/isoelectricpoint

You can use this charge to get it to stick to an oppositely-charged column, then alter the pH to get that charge to change or add a bunch of salt (which is charged) to push it off. But lots of proteins have similar charges, so this technique is good for a “second step” column once you’ve removed most contaminants by using a more specific property. more here: http://bit.ly/ionexchangechromatography

Thankfully, in addition to exploiting proteins’ natural different properties, if we’re using recombinantly expressed proteins, where we put the gene in cells that don’t normally have them and make them make our protein, we can alter them to have really different properties by adding an affinity tag that binds to the specific placards on the museum walls (functional groups stuck (conjugated) to the surface of the beads, letting your protein stick, then competing it off with something that looks like the tag. more here: http://bit.ly/streptag & http://bit.ly/histagpurification

Even if the proteins find the museum really boring, they’re forced to go through. And in a different kind of chromatography, SEC you want the museum to be as boring as possible because you want all the proteins to be traveling the same rate – but traveling different distances. 

The SEC beads have “secret tunnels” that are like exhibition rooms. But they only let in people who can fit through the door. So little kids (small proteins) go through a lot of exhibits and thus have farther to travel then big people (big proteins) or groups of proteins (proteins stably bound to one-another) so small proteins take longer and exit later. more here: http://bit.ly/sizeexclusionchromatography

This is the opposite of what you see with SDS-PAGE. There, smaller things travel faster so when you turn off the electricity they’re trapped lower down in the gel. This is because, PAGE is like an audio-guided museum tour. You follow a set route, but go at your own pace (bigger proteins get tangled up in the gel mesh more) whereas SEC is like a “docent-led” tour where your size is like a “docent” that determines which path you take and therefore how fast you’ll travel. more here: http://bit.ly/sdspageruler

For more practical protein-purification posts (and background/theory), check out the page on my blog where I’ve collected some of my protein purification posts. http://bit.ly/proteinpurificationtech


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