Centrifugal ultrafiltration (spin concentrators) for protein (or nucleic acid) concentrating
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- เผยแพร่เมื่อ 1 ส.ค. 2024
- Some days, I find it really hard to concentrate… my protein that is! Centrifugal filters (spin concentrators) to the rescue! “Centrifugal ultrafiltration” which is just a fancy-dancey 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.)
blog form (also has static pics): 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 the MWCO are pulled through the membrane into the lower (waste) chamber, but molecules bigger than the 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 1/2 the size of smallest thing you want to keep. Note: this might remind you of dialysis… 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
for details: bit.ly/spinconcentrators - วิทยาศาสตร์และเทคโนโลยี
Honestly amazed how you keep coming up with your topics. Wish this channel was around when I studied Biochem. Awesome resource! Keep up the great content!
10k watching, thanks for your introduction🦋,love from China
Thanks! Happy to help. Good luck!
Greetings from Brazil. Great video. I'm going to perform this centrifugation for the first time and your video helped me a lot.
Thanks! Glad to hear it!
This was great, thank you so much! Helped me a lot when I was asked to use them in a protocol for the first time!
So happy to hear - thanks!
Thank you so much!
Thanks!
i love your videoas and explaination, they are really helpful always for my lab work😍
So glad they're helpful!
thanks :D
Hi! Thank you so much for sharing. It's super helpful. I'm wondering what speed you use for the swinging-bucket centrifuge?
Great to hear it was helpful! You can go up to 4000 g with these
Hi! Thank you for all the tips and info about these devices! When reusing the Amicon 15mL (that looks like a 50mL falcon), what volume of a 20% ethanol solution should I add? 5mL is enough? Also, at what temperature should I store these devices between uses? Thanks
I'd just make sure there's enough liquid to keep it wet. And I kept in fridge.
Very much helpful. May you please assist, i'm need to separate laccase, manganese peroxidase and lignin peroxidase from crude extract based on size exclusion. Which column and buffers would be ideal for this experiment?
Glad you found it helpful! I don't have experience with those specific things so I recommend you try to find some published protocols for it. Sorry!
Thanks for sharing another useful video. I usually go through some of your content before doing "simple" stuff like Transformation or Purification.
One small question : Lets say that I am using a Centrifugal concentrator with a 10kD cut-off. After adding the protein-containing solution to the tube, do you know of a method to estimate the optimal speed at which the protein in the tube would get concentrated in the least possible time ?
P.S. Based on some of my internships, I learned that the estimating the optimal speed at which a protein can get concentrated is usually determined by a trial and error basis. But I was curious if there was something I was missing.
- Bart
Glad you found it helpful! The manufacturesrs' website should have guides for that I think. Good luck!
have to mix them often. there gets to be a large concentration gradient some proteins will begin to crash (precipitate)
👍
Hello, with what solvent should I wash the filters to reuse them?
I think I used to use 20% EtOH
Hi! Thank you so much for sharing. I have a problem at this step in my experiment. I need collect MWs
I don't understand what you're asking sorry but good luck!
That mean, ~200mg protein hydrolysate. What volume of 20% Ethanol I should add? From 10mL enough?
I don’t know to do centrifugal ultrafiltration that is there a concentration for sample solution?