Hello.
I am trying to separate three different proteins (350 kDa, 240 kDa, 64 kDa) via gel filtration chromatography, and this is the first time for me.
In my investigation, sephacryl 200-HR is good to separte proteins.
(Guideline says Sephacryl 200-HR is used for globular proteins under MW range of 5~250kDa).
For the separation, 240 kDa of proteins have to penetrate into 250 kDa pores of resin..
But, I am wondering if 240 kDa of protein will penetrate into 250 kDa pores of resin effectively.. too TIGHT!! How do you think?
Can you recommend best column resin and glass column size (diameter and length) ?
Any comments would be helpful to me. Thank you.
Hi,
A Superdex 200 would give you better resolution than a Sephacryl. Other factors for improving the resolution are the length of the column vs. the diameter (the longer the better), and the sample volume vs. the column volume (the smaller sample volume the better).
However, regardless of the column used achieving good separation of the 350 kDa and 240 kDa proteins maybe be challenging. As a rule of thumb one can obtain baseline separation when there is a two fold difference or more in molecular weight.
Nonetheless, don't forget that the way that proteins migrate on a gel filtration is dependent on their molecular size and shape (Stokes radius). Therefore, it's always necessary to test where your proteins elute, unless you're sure that they're all globular (or have other prior knowledge of the shape).
The most useful resource that GE offer is the size exclusion handbook (http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc18102218_20140818002953.pdf). This is a little dated, but broadly is an excellent guide to the method. Page 16 has a very helpful table.
In your case, the real question is what amount of protein you are looking to purify, and the volume that your protein will be in. This significantly affects the answer. In general, small quantities of protein (in mg terms) tend to get "lost" on size exclusion columns that are too large. As a rule of thumb, if you have 100-1000 ug of each protein, then a smaller (e.g. 24 mL) column will be suitable, aiming for a maximum sample volume of 0.5 mL; for a greater sample, then a bigger column is likely to offer better resolution. You can buy 120 mL columns pre-packed which are suitable for samples of 2 mL in your case. Your sample volumes will need to be smaller than usual (normally maximum 3-4% of column volume is recommended) because you are trying to separate two proteins that are reasonably close in size.
Your other question is what resources you have available. The key questions are:
- Do you have a chromatography system?
- Do you have a budget to buy a column?
It is possible to run a gel filtration by gravity flow, using a Sephacryl resin. However, the resolution is pretty poor on these. You will probably be able to separate your smaller sample from the other two, but there is no chance of separating the larger two. Much better is to use some sort of chromatography system (e.g. AKTA series, but others can be good). Your institute will almost certainly have something suitable. However, you need to check the specification, as the pressure limit on some low-end systems will not support all of these columns.
Buying a pre-packed column is _expensive_. List price for Superdex columns start at £1338 in the UK. However, they give excellent performance, and, properly looked after, will give several hundred runs. Packing your own is not for the faint-hearted. I couldn't find a good video on Youtube. If you want to pack your own (i.e. the pre-packed is too expensive!) it might be better to go for Superose. The resolution is a bit poorer, but the packing is more straightforward, and the columns run at lower pressure. For size exclusion, you want a long, narrow column, as separation is much better in these. The column sizes for the pre-packed columns are a good guide for what you would want.
Finally, when it comes to running the column, I suggest that you read the manual above carefully. The main point is that size exclusion is supposed to be run fairly slowly - you want to set the flow rate so that the column is running at a maximum rate of 30 cm/hour (i.e. if your total column length is 60 cm, the column should run no faster than two hours). You can improve the resolution if necessary by dropping to 5-15 cm/hour.
Good luck!
Hi,
IF you use Sephacryl s-200, 240 kDa protein maybe not be isolated from 350 kDa protein, both of them will probably appear in the breakthrough peak,so you can try Sephacryl 300, but it is nearly impossible to separate these three proteins well in same one colume. Of course, the resolution will depend on the loaded ampunt of sample, the length of colume, and flow rate,a and so on.
Hi,
If Sephacryl s-300 was used, I am afraid more bad resolution will be get.
You should use resin that includes all the molecular weights of your desired proteins in its separation range. From my experience, if you use a max. 250 kDa resin to separate yours proteins including a 350 kDa protein, it will be too large to go through the labyrinth, and eventually you won't get your largest (350 kDa) protein.
The smaller the loading volume the better, and the longer your column is, the better separation efficiency it is. High efficiency columns also consist of smaller diameters, despite significantly low flow rate (but can be increased by air/vacuum pumps). For my previous experiment I manipulated on Sephadex G-100 (4~100+ kDa) to separate my ~65 kDa protein from the pool. I used a 25 x 5 cm glass column, which was relatively short but it was fine as I only required to purify one protein from the rest.
You can refer to the journal I attached. Hope it helps. Thanks :)
I don't get the above link to the GE Healthcare gel filtration handbook to work.
Try this link to the download page: http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/sv/GELifeSciences-se/service-and-support/handbooks
you can go with Toyopearl resin also to seperate the compound by Gel premeation chromatography
Hi Sung,
After many years of using prepacked SE columns (both GE Healthcare and Bio-Rad are nice, but expensive...) I have swithed to self made. I have fallen i love with Bio-Works media. They are realy fast flow and high preasure resistant, thus all the washing and equilibrating steps are 4x fasten. The price if you have the column to pack is 20-40x cheaper is giving you the freedom of runing crude extracts can stuck the column. WorkBeads 40 SEC with separation range 50-1200 kDa I find as suituable for your experiment.
best - jan
Hi all,
I am also trying to separate my protein of interest which is of 35 kda from rest of the impurities which is of 20 kda, 30 kda by using superdex 75 10/30 column on fplc sysyem. However i am not able to resolve the proteins. in one single peak i am getting two proteins. the volume of protein i load is 200 ul and the flow rate i maintain is 0.75 ml /min. but still not able to resolve.
kindly consider d issue and please let me know how to get it resolved. Hope for your kind reply soon.
To see if the SEC separation of the 20, 30 and 35 kDa proteins is at all possible (the smaller protein MAY have an elongated shape that makes it behave as a somewhat larger protein !), make a test run with very much slower flow rate than 0.75 ml/min. E.g., an overnight run with 0.1 or 0.05 ml/min.
But first: For best resolution you should see to that you use as small extra-column volumes (dead volumes) as possible, i.e. mount column directly between sample injector and UV-cell, with as short and narrow tubings as possible. Likewise, from UV-cell to fraction collector. Do not use pH-cell (too large dead volume). CAUTION CAUTION: if skipping the column valve to minimize dead volumes, you may have to disconnect the column when doing pump wash, system wash or other high-flow operations, so that the high flow by mistake does not enter the column and compress the packed bed.
Can anybody help me that which column I should use in place of Hiload Superdex 16/60 200pg for histone core octamer purification.
My Protein is Cadmium binding and around 25 KDa molecular weight. PI is 4. 76. Can anybody recommend me which column and chromatography should I use for protein purification. Its soluble protein. I have taken its expression in ammonium sulphate precipitation and Dialysis also. Kindly guide me about its purification.
Hi Asmara, For this protein purification you can try Ion exchange chromatography again column selection either Cation exchange or Anion exchange depends upon you protein of interest.
All the best
Can anyone suggest me column length and volume of sample to use for plant protein extracted in lkaline media.
I have a diluted sample of 200ml which contains very low protein content and i want to run it on Sepabeads 825L; can anyone suggest me the protocol to how to go about it regarding regeneration of resin, sample volume, eluent flow rate?
- Badges
- Science method