Practical tips for using spin concentrators (centrifugal filters), esp. for protein concentrating скачать в хорошем качестве

Practical tips for using spin concentrators (centrifugal filters), esp. for protein concentrating

“Centrifugal ultrafiltration” which is just a fancy-dancey-hard-to-say-way of saying you stick your too-watery protein solution into a membrane-lined tube insert and spin it really fast. The force from the spinning pulls the water (plus salts and other small things) through the membrane, but your protein’s too big to get through the membrane’s pores so it stays put. Sounds pretty boring - and it is - especially when your protein is taking hours to concentrate to the desired concentration… but it’s really important and we do it a lot so today’s a practical, post I hope will bore you not…Some details on the what and the how and then some of the why’s (preparing for SEC, preparing to freeze, buffer exchange, etc.)    text from blog form: https://bit.ly/spinconcentrators Protein concentrators come in many volume-holding-capactities (e.g. 0.5mL, 4mL, 15mL) & molecular weight cut-offs (MWCO) (e.g. 3K, 5K, 10K, 50K). MWCO refers (indirectly) to the size of the membrane’s pores. It’s 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 top). 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.     You put your sample in the top chamber & spin it it the centrifuge.   Molecules smaller than MWCO are pulled through the membrane into the lower (waste) chamber, but molecules bigger than MWCO stay in the upper chamber     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.” So to avoid losing protein, you typically choose a MWCO 1/3 the size of smallest thing you want to keep. Note: this might remind you of dialysis… https://bit.ly/proteindialysis    Another important thing to keep in mind is that, since it’s an average pore size and since all the proteins are still able to mix around with one another, it’s NOT useful for separating proteins by size. Ultrafiltration can only be used to separate things that differ by a magnitude of size. So I can separate my protein from salts, but not from another protein.     Also, since we’re on the topic of salts, you can use this as a way to “desalt” a protein and/or switch it into a different buffer - concentrate the protein and then re-dilute it in the buffer you want.     I usually concentrate it in spurts of 15min or so depending on how much concentrating I need to do. In between spurts I use a pipet to mix around the liquid, especially near the membrane, where gunk can build up on the membrane walls and make passage more difficult.     There are a couple of times during the protein purification process when you want/need to concentrate your protein  1. before Size Exclusion Chromatography (SEC) (aka Gel Filtration)  2. before freezing your final product    Why before SEC?    Protein purification usually involves a technique called column chromatography, where you pass a solution containing your protein of interest (and other proteins) through a series of columns filled with little beads called resin that have different properties and interact differently with different proteins (because different proteins also have different properties), allowing you to separate proteins by things like charge (with ion exchange chromatography) & size (with Size Exclusion Chromatography (SEC) and isolate the protein you want. http://bit.ly/proteincleaning     A lot of forms of chromatography in effect concentrate your protein for you. Take, for example, affinity chromatography, where the resin is specifically sticky for something special about your protein. Yesterday we looked at one such form, where I had a His-tag on the end of my protein which binds to nickel bound to the resin. I used this technique again this morning. When I added a “dirty” protein mix, my his-tagged protein stuck, the other stuff flowed through, and then I pushed my protein off (eluted it) with a competitor called imidazole. http://bit.ly/histagpurification   Finished in comments