Interpreting & working with protein chromatography chromatograms - w/practical look on an AKTA

Protein chromatography is a way to separate proteins based on differences in their properties - size, charge, etc. by flowing them through columns filled with little beads (resin) that interact differently with them based on those properties. We often do this with Fast Protein Liquid Chromatography (FPLC). Here a machine like the AKTA takes our protein sample and pumps it onto a column (which it’s gotten ready by flowing a bunch of buffer (pH-stable salt water) to “equilibrate” it. It then washes the column with the buffers we tell it to. And at the speeds we tell it to (FLOW RATE). When our protein comes off of the column (elutes) we see it as a peak on a UV chromatogram. So today, here’s a look at how to interpret then and a practical look at working with them on an AKTA. Including some of the “extra” things to add to the curve to monitor - like conductivity & pressure. blog form: https://bit.ly/chromatograms

Here’s a little text from a past post, but the video’s the main thing today because it’s easiest to see with examples!

much more on chromatography here: http://bit.ly/proteincleaning

The liquid flows through lots of little tubes that offer different FLOW PATHS. Which path the liquid takes is dictated by lots and lots of valves to go with those lots and lots of little tubes. It’s kinda like a subway system that can change the tracks. So we can direct liquid into different columns and, when it comes out of the columns into the waste or a fractionater which collects it to “keep.”

It can collect different fraction sizes
for things like the flow-through in affinity chromatography, where your protein shouldn’t be there, you mainly collect the flow-through as a “just in case” so you don’t want to collect tons of tiny fractions, so we usually collect things like that in 50mL tubes
if you have a LOT of flow-through (like the other day when I was trying to get my protein to bind an anion exchange column but there was too much salt so I had to super dilute it and ended up having to flow almost 2L through a 5mL column) you can direct it through an “out” valve hooked up to a tube you can stick anywhere - like a giant graduated cylinder
when we’re doing the actual protein elution, we want to collect smaller portions for a couple reasons
1) because the more concentrated it is the easier our life will be (and, as long as it’s not so concentrated that it clumps up (aggregates), proteins are usually happier when they’re more concentrated, so the next step is usually concentrating the eluted protein and the more concentrated it is to begin with, the less concentrating we’ll have to do
-for things like stepwise elutions, where you unstick everything in one fell swoop (e.g. go from no competitor to maximum competitor) 15mL tubes are good for this
2) if proteins come off the column at different times but you collect everything that comes out in one tube, you’ve unpurified what you purified! But if you collect smaller portions, and different things are in different portions, you can keep separate the things you separate!
for gradient elutions (work your way up to that max competitor concentration) depending on how tightly your protein’s stuck on, it’ll elute (get pushed off) at different concentrations - we often collect the elations in 96-well blocks. Each well can hold up to 2mL, but you can program the AKTA to do smaller fractions than that so you can be really picky about what you want to keep

We choose which fractions we actually want to keep based on the CHROMATOGRAM. This is where we see the evidence of our protein coming out in the form of a peak in the 280nM wavelength absorbance. Proteins (in particular tryptophan, tyrosine, and phenylalanine) absorb that type of light so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. A UV MONITOR on the path between the bottom of the column & the fractionater measures this. And the computer shows this to us as a peak.

We have 2 SYSTEM PUMPS so you can use 2 different buffers that send liquid first into a mixing chamber so you can mix them if you want to make a gradient for a gradient elution to introduce the “competitor” that will push your protein off the column (e.g. have a no salt & a high salt or a no imidazole & high imidazole (for His tags) you can mix). Or you can just use 1 for an “isocratic elution” like for SEC when you don’t need to change the buffer.

Another thing it has is a CONDUCTIVITY MONITOR to follow column equilibration (you want the conductivity to be stable before you put your sample on - this indicates that all the liquid in the column is the one you want your protein to meet) & salt gradient formation/ We have a pH MONITOR too.

more on the AKTA: blog form: https://bit.ly/aktainaction ; video:    • Meet the AKTA! (and don't be intimidated b...    

protein UV absorbance: http://bit.ly/bradforduv

more: https://thebumblingbiochemist.com/let...