I’m a desalting column, short and stout – small things stay in while bigger things flow out. The reason they do this is that they take a shorter route, so put your sample in and exchange the buffers out! It’s not just proteins I use chromatography for – this tiny little column may look like a miniprep column but it’s actually a tiny little size exclusion column I use to separate excess label from the RNA I radioactively label. But that’s not all desalting columns are useful for – and they’re not all this small. We can use bigger versions for proteins too and use them to remove excess salt or exchange the liquid our sample’s in). Let’s take a look starting a way you probably won’t find in a book…
note: text from 2019, video & some graphics new
Imagination you have a turtle race with a crowd of turtles en route and then partway through the race a cheetah and a crowd of tortoises come in. The cheetah’s gonna beat all those tortoises, but since the turtles had such a head start, it’ll finish in a crowd of them. And if you stop the race before the tortoises come in you get that cheetah in a crowd of turtles (the starting racers) instead of in a crowd of tortoises (the partners it came in with).
Now imagine that, instead of a cheetah, you have one of those giant tortoises which are so awesome. They don’t move faster than the turtles, but they get to take a shortcut. They’ll still finish ahead of the turtles they come in with that don’t get to take the shortcut, but they’ll finish with the starting crowd. This is the concept behind buffer exchange
You have a column filled with resin (little beads) and those bead have “secret tunnels” – winding pores that only molecules small enough to enter them can go through. Molecules (be they proteins, DNA, RNA, etc.) that can’t fit get to go around (shortcut!) but molecules that can fit (like salts, free ATP, etc.) have to go the long way. And because they have to travel further, they take longer to go through.
Before I talked about how I use size exclusion chromatography (SEC) during protein purification as a “polishing step” to separate the protein I want from proteins that are different sizes. In that case, I want good resolution (ability to separate similar-sized things) – so I want a “long racecourse” so that the molecules encounter more beads, with smaller things getting slowed down more and more and you can catch the outgoing molecules on their way out by taking fractions. If you want good resolution, you want a long, skinny column with little beads
But if you want more of a yes/no resolution (like salt vs protein) you want a short, fat column and big beads.
This G-25 microspin column may look like a miniprep column, but it acts like a size exclusion column. “Miniprep” & PCR Purification spin columns are also little columns. The only reason that these desalting columns look like those is because they’re all designed to fit in a microcentrifuge
When you put DNA through a minipreps or PCR purification columns to purify DNA, you add it under conditions where the DNA will stick to the membrane. Then you wash the other gunk through and then you change the conditions so the DNA will come off.
With these desalting columns, you’re not changing any conditions and the DNA is NOT sticking – nothing should really be sticking it’s just that you don’t give the slow stuff enough time to travel through the column and it can be used for “buffer exchange” because whatever is currently in the column will come out with the big thing I put in.
I use these G-25 microspin columns when I radiolabel RNA to remove the excess hot ATP. Because of the weird backwards-writing nature of chemical synthesis of oligonucleotides (they’re written 3’ to 5’ with the OH on the 5’ arm instead of the 3’ leg), you’re left with a free 5’ OH instead of a free 5’ phosphate. if you want to make it “normal” you have to add a phosphate. You could just add a normal phosphate, or you could add a radioactive one so you can track it
Phosphate-adders are called kinases and for 5’ end labeling I use a kinase from a bacteria-infecting virus (phage) called T4 – T4 polynucleotide kinase (T4 PNK) to do this. It adds the phosphate from ATP (adenosine triphosphate) and you can give it radioactive ATP to use. Just add T4 PNK, hot ATP, the right reaction conditions, etc. & wait – you put in a lot more hot ATP than you need so you want to separate it from the ATP that’s attached to the RNA. We use easytide ATP which has a green dye in it and that dye should stay in the column
The G-25 is the name of the resin, which is a form of “Superdex” – the dex is for dextran – a sugar that, along with agarose, forms the beads’ gel mesh. These versions are small, perfect for the small volumes I’m using, but they also have bigger ones, including ones you can use for proteins. In undergrad, I used PD-10 columns for buffer exchange of proteins – those are just bigger versions of these little guys
The DESALTING part comes in handy for things like ion exchange chromatography (IEX), where you get a protein to bind to a column based opposite-charge attractions, then you add increasing levels of salt to outcompete it (when a salt dissolves it breaks into its component ions (charged particles) – e.g. table salt (sodium chloride, NaCl) becomes Na⁺ + Cl⁻. This can leave you with a very salty (though hopefully also very pure) protein.
To remove that excess salt you could use dialysis, where you put the protein in a semipermeable-membraned pouch and put it in a bunch of low salt buffer – protein stays in the pouch & salt flows out until concentrations of salt equalize inside & out. Cheap & does the trick, but takes a while… you can instead use a desalting column which you “pre-fill” with the liquid that you want your protein to come out with. Also good for removing small competitors like imidazole that you might use for affinity chromatography.
more on other size exclusion chromatography resins: http://bit.ly/sizeexclusionchromatography
more on dialysis: https://bit.ly/proteindialysis
more on protein chromatography: http://bit.ly/proteincleaning
more on radiolabeling: http://bit.ly/radiolabelings
more on oligo synthesis: http://bit.ly/2We8e8W