Centrifugal ultrafiltration - concentrating protein using spin concentrators (e.g. Amicon)
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- เผยแพร่เมื่อ 7 ก.ค. 2021
- 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/2KxDEVF
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
This is cool! I'm doing purification and buffer exchange so this is helpful. I appreciate the details in the description and the images on the site
Thanks - glad it was helpful! And best of luck with the experiment
This was SO helpful! thank you very much!
I'm purifying some protein as well and your vids have become so helpful. Thank you
So glad to hear! Good luck!
Hey, I work in the department that makes these, it's a whole Centrifugals department, so you'd get plenty of practice in pronunciation 😄
These are all made in Cork, Ireland, was looking for a video to show the team the end use of the devices so was delighted to find this.
Keep sciencing, and sharing the knowledge.
oh awesome! thank you!
Thank you for the information.... great video 👍
thank you!
Thank You for the video. We usually store it in 20% Ethanol.
This video is so useful for me right now. Thank you so much. I wanna use them multiple times too, how concentrated is the ethanol you use for storage? How do you remove it before using it again?
Glad to hear it! I don't remember exactly what I stored it in, but if you do a lot of buffer washes it should be good to go. Good luck!
Thank you for this video, was very well explained and easy to understand. I wanted to ask you, do I need to calculate my protein concentration by an assay (for ex. Bradford Assay) before using the centrifugal filter?
Only if you're curious and/or if you're aiming for a specific concentration. And thanks!
Thanks for the wonderful video! If I wanted the final volume to be 100-200 uL using the 50 mL tube size, then some part of the membrane would become dry during the centrifuging, would this cause my protein precipitate on the membrane? What would be a better way to do this? :)
Glad you found it helpful! The protein should actually all concentrate at the bottom - it should get pulled down and therefore not get stuck on a dry membrane (but you still do have to worry about protein sticking to the membrane in general)
Hlo ... Can u plz tell by which material this memberane is made up of and why only that material is used as a memb instead of any other one ???
They're typically regenerated cellulose chosen because they're hydrophilic and highly compatible with lots of different things and have low protein binding www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/protein-concentration-and-buffer-exchange/protein-sample-ultrafiltration
Hey thank you for your video! It was very helpful. I am kinda new at this and I am lost with the preparation of the tubes with resin and buffer. Any resources or tips about it? Is it needed even for concentration?
Glad I could help! You shouldn't have any resin when you're concentrating! Are you thinking of chromatography?
@@thebumblingbiochemist no I'm not! Okay great. I thought that would be the case from what I've read but I was not totally sure. Thank you so much :D
Hi. May I know how many percent of Ethanol are you using to store the centrifugal tubes? And What temperature you store it? For example using the Amicon ultra 15, how many mL will you pour the Ethanol?
20% ethanol, enough to make sure the whole membrane stays wet, and 4°C
Hi, can i ask whether you can do overnight dialysis at 4 degree celcius with the protein concentrator tube?
These tubes aren't designed for dialysis - instead you'd want to use a dialysis pouch
I wonder how well these will do with lipid nanoparticles. . .
really fine ones. Like 30 nm in size... tried super fast centrifuging like 50k Gs for 30 minutes... didn't work so well.
never tried but maybe www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/filtration/nanoparticle-ultrafiltration
@@thebumblingbiochemist thanks. I though 50 kda would would looks like I miss read.
-30 would be better. My particles reach 11-12nm at the minimum. So very tiny...
good luck!
Regarding using Amicon 100 KDa, etc. I found a protocol using 3000 g for 5 minutes. My centrifuge can do only 1400 g. Is it possible to do ultrafiltration in this conditions ? You have a practice experience.
It will probably just take longer
@@thebumblingbiochemist m
Many thanks.
Hi, If I wash with MeOH 100%, can I use again with other sample?
I've used ethanol in the past - best to use with the same protein if possible