Don’t cut *me* off, but when performing DIALYSIS, it’s OK to cut off molecules! (as long as you choose the right molecular weight cut off (MWCO)…) Sometimes, to purify protein, we have to first swap out the “dirt.” BUT then we can use dialysis to “clean up” our new mess. Here’s how it works 

note: text adapted from several years ago, pre-blog days, video’s new & has more details than I have in text, cuz – well, if you saw my lab bench today you’d understand…  

There are lots of times you might want to use dialysis during a protein purification. Sometimes after affinity chromatography. In that technique, we flow solution containing our protein (& other contaminating proteins) through a resin column (column filled with little beads). Our protein sticks, BUT other proteins flow through. This separates them (yay!), BUT now our protein’s stuck to resin (boo!).

To get it off (ELUTE it), we need to flow through something that makes our protein less attracted to the resin or competes for it. This kicks our protein off (yay!), BUT now we have some of those small molecule “somethings” in our eluted protein that can cause problems (boo!).

Thankfully we can remove them by DIALYSIS, a technique that harnesses the power of DIFFUSION (random movement of molecules that leads to a net movement from areas of high concentration to areas of low concentration, leading to an evenly mixed solution). More on diffusion here: 

In DIALYSIS, we use diffusion across a SEMIPERMEABLE membrane (only lets certain molecules through) & we choose the membrane based on which molecules we want those to be.

BASIC CONCEPT: put protein in membrane pouch ⏩ put pouch in big bath that doesn’t have that thing we’re trying to get rid of (we call this bath liquid the DIALYSATE) ⏩ If the membrane’s permeable to that thing, it’ll leave pouch & enter bath, but our protein can’t get through the membrane, so will stay put.

At the molecular level, molecules are randomly moving around within pouch & will “bang into” membrane. If it “bangs into” a place where there’s no membrane (a pore)(& is small enough to go through) it’ll go through.

BUT it can also come back in because the same thing’s happening on the other side of the membrane, so you want volume (V) of bath to be MUCH greater (usually ~200-500X V of pouch). This way, once thing exits pouch, it moves far away, where there’s less chance of banging into membrane & coming back in.

BUT eventually concentrations of thing inside & outside pouch even out – you’ll have equal concentrations of thing on either side, so you’ll have equal movement in both directions. You’ve reached a DYNAMIC EQUILIBRIUM.

BUT you still have that thing you don’t want in the pouch (though there’s less of it). How much less? It depends on initial volume ratio. For example, if you start w/1:200 ratio (e.g. 1mL sample in 200mL bath) you’ll end with 200-fold decrease.

To get rid of more, you need to get back to NON-EQUILIBRIUM. You can do this by swapping out the bath liquid with fresh bath. Now you have a concentration gradient again & thing will move out…

… Until it reaches equilibrium again. Ugh… But now you have even less of the thing (e.g. 200 x 200 = 20,000-fold decrease) & you can keep doing this until the thing is almost completely gone

This can be a slow process. You can speed it up by increasing temperature (adding heat gives the molecules more energy to move) BUT this can be harmful to your proteins. A “safer” option is to increase the surface area to volume ratio of pouch so more molecules come in contact w/membrane & have a chance of escaping.

This is strategy behind these dialysis “cassettes” which have a thin rectangular shape. They come in a variety of pouch sizes (choose depending on V of protein solution) & MOLECULAR WEIGHT CUTOFFS (MWCOs) (choose depending on size of thing(s) you want to get rid of & size of the thing(s) you want to keep)

MWCO is given in units of Daltons (Da) & tells you molecules below this size can go through (are penetrating) but molecules above this size are retained (are non-penetrating & stay in the pouch)

You want to choose a MWCO smaller than your protein (& anything else you want to keep) but larger than whatever you want to get rid of. 

The bigger the pore size, the faster you’ll reach equilibrium (because if a molecule bumps into the membrane it’s more likely to “bump into” an open space it can get through & doesn’t have to worry as much about “squeezing” through. BUT you want to be careful not to select a size too close to your protein size since the MWCO is an average, so you still might have pores big enough to let your protein through. 

Typically, a MWCO “guarantees” that at least 90% of molecules of that size will be retained. BUT proteins have different shapes which MW doesn’t account for (e.g. a long skinny protein might be able to “slither through.” To avoid losing protein, you typically choose a MWCO 1/2 the size of smallest thing you want to keep

note: another place we see MWCO come up is with ultrafiltration membranes in spin concentrators we use to concentrate proteins: 

Speaking of concentrating… In addition to de-salting & buffer exchange, you can use dialysis to concentrate proteins if you put a high concentration of non-penetrating solute (like big PEG molecules) in your bath. This makes the bath hypertonic compared to the pouch, so water flows out. But typically, we’re more worried about too much water flowing in since things are typically more concentrated particle-wise in the pouch. More on osmosis & tonicity here: (another super-old, pre-blog post I will have to refresh & post at some point)

Final note: HEMODIALYSIS uses a similar process to artificially remove waste products from the blood of patients with kidney problems. Blood’s removed & passed through an artificial kidney (dialyzer) in which it’s separated from a waste-free dialysate by a semipermeable membrane that doesn’t let the blood cells or proteins through, but does let through small waste products like urea & creatinine. Therefore, waste products diffuse out & clean blood’s returned to patient. And speaking of creatinine: 

more posts on purification: 

Leave a Reply

Your email address will not be published.