Perform serial dialysis of your sample

@Ranjan, I've tried serial dialysis, and unfortunately I lose ~50% of my protein sample (as determined by BCA assay), and even though the dialysis was done at 4*C, I'm worried that my protein may begin degrading during the rather long dialysis process. I should have included that in my question, and have now added it.

You have plenty of options.

First are the spin columns. The ones from Pierce are perfectly fine and should remove most of the salt. You just have to add your salt-free buffer repeatedly.

The other option are dialysis or ultrafiltration, but these take rather long time. Do not dialyze against water! Always use buffer!!!

Last, you can use desalting chromatography columns. Your link didn't work, but if I found it correctly, it was spin column as well. Anyway, the desalting chromatography works well and is fast, but your sample will be slightly diluted. But remember you should not overload your column to achieve sufficient desalting!

Use gel filtration column (Ex . 25 ml Superdex 200 ). Use a buffer (without salt ) as running buffer. This will surely remove 98 % of the salt content. Your protein will be diluted but you can concentrate it with centrifugal concentrator.Also once you concentrate it then again dilute with the running buffer (No Salt) and perform the concentration step again and this will remove the left over salt too.. you can perform the later step 2-3 times for surety. the overall process will take 3 to 4 hours and i am sure it will work.

Prof Zoran; what is the problem in using superdex 200 column? It works ok in the given MW range very well. Of course if she does not have that column then buying that column is not a wise decision and then she can go with whatever she has (provided MW range works). Superdex 200 is just an example. she can use whatever she can. Longer the column more is the dilution and more will be the buffer exchange and thats all i want to point out. Well if she does not have FPLC in lab then she probably needs to use any standard desalting column.

BCA assay if wisely used is good enough for comparing protein loss ( it wont be accurate, but its a good estimation) and i assumed she will estimate protein using BCA for both before (control) and after dialysis (keeping in mind that she dilute the sample good enough so that salt do not interfere). according to her there is 50% protein loss and kind of current protein estimation method will for for knowing that kind of loss. or if you are really worried then just measure the absorbance at A280 and take the ratio for both before and after dilaysis but of course there is no point of doing all these for her current research interest so just move on.

as prof. Vujcic wrote, Superdex 200 is not suitable for buffer exchange. If Lauren wants only to exchange buffer and not further purify her protein, she would end up with several tens-times diluted sample and she'd had to concentrate it anyway. Thus any gel permeation matrix with low resolution such as Sephadex G25 or G50 are much better, because all the proteins will be in only one peak.

As some people already mentioned. Sephadex G25 is ideal for desalting protein solutions. I normally use the PD10 desalting columns from GE: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdoc52130800BB_20110830191706.pdf

This gives you a desalting capacity of 90-99% while diluting 2.5mL to 3.5mL. The only limitation is that you need to start with exactly 2.5mL either by diluting or concentrating your sample.

You can use a little tube of cellulose acetate,fill with your solution for half of his volume,put tube in demi water for one day or more.This is an old easy method for desalting.

First, you have to read a book for basic biochemical research techniques , Then you have to test is your protein is stable a lower salt concentrations. Once this is done the simplest method to reduce concentration of your protein is to use a desalting column or simple dyalisis. This is the most innocuous for the protein even though some aggregation or precipitation can occurs. Other possibility is to concentrate your sample and then dilute it with your new buffer. This concentration has to be repeated twice more in order to be sure about the final concentration. I do not recommend precipitation with acetone because depending of how much your protein is resistant you can lost massive amounts of protein.

Any precipitation with solvent might damage your protein, column desalting is a good one but for small amount of protein because capacity of such type column is very low.

I usually do UF/DF that is close to dialysis, to do UF/DF you need to get membrane set and typically to prevent losses the pore size should be 2-3 times lower your protein size, therefore if you have 50 KDa then your membrane should be 15-25 KDa as cut off.

I think you can find easy how to do UF/DF, and in short words you do UF = tangential UF then add your new solution without salt to the same volume, and repeat it 10-20 volume of your initial solution. It can be done also as continues adding your new solution and removing your old one as a filtrate.

To set up it you need peristaltic pump with flow rate as high as possible, silicone tubings, pressure gage. Typical time after setting up is 2-4 hrs.

Prof Zoran Vujcic ·Agreed what you said

I think using the centrifugal filter devices is one of the best way for desalting of the protein samples.

for preparing samples for 2d page, i always follow methanol-chloroform precipitation. it is fast, can be done at RT and recovery is good (also depends on ur working hand). u can find the detailed protocol on google also. i have also attached two files. this is reliable protocol, so dont worry, just go for it.

Here is the second protocol. u can also follow this paper for the protocol (http://www.ncbi.nlm.nih.gov/pubmed/6731838)

I too use Zeba spin or GE PD-10 desalting column depending on volume of sample after equilibrating them with water/buffer of choice, they work for me.

As I know in 2-DE you will have to use hard denaturing conditions such as 9M Urea and 10 mM DTT, Also it is imbprtant for you that have a concentrated sample of potein. So I also think you could simply precipitete your proteins with cold acetone or even TCA protocol.

My suggestion is to use Millipore Centricon filter devices as they are very simple and easy to use with quick results. They are suitable for small volumes and available at 10kDa, 3kDa and other sizes too. Simply apply the sample into the top sample reservoir and centrifuge it at recommended speed setting for 30mins or so. You can keep the filterate and check for any proteins just in case if membrane is damaged or not the correct pore size. You have to top up the reservoir witht the required buffer three or four times to achieve the best results. Recovery is good as well. I don't have to explain every thing as the short instruction sheet can give precise information.

Hi Lauren. First of all let me ask you a question about the pI of your protein. What is the pI of your protein. For e.g. if I have a pI of protein lets say as 6 then a buffer of 50mM Phosphate (either using K-salt or Na-salt) pH 7.0 is good to be dialysed in. Depending on the volume of your protein sample you will have to work the volume of this reservoir buffer needed to remove majority or all of your salt. You can use dialysis tubings of certain mol. weight cut offs like http://www.piercenet.com/browse.cfm?fldID=FEEDDA2D-AE4F-29E9-F4D8-986E921E193B. Here you can fill this tubing with your protein and dialyse it in the cold room overnight at 4 deg. C or room temperature for 2-4 hours depending on how happy your protein is at room temperature. I usually stick to the cold temperature condition overnight duration. I usually elute my protein on affinity column using 150mM NaCl and then remove all of the salt once purified by dialysis in this tubing. Your sample will get diluted to a certain extent as you will be losing out the NaCl solute molecules. Hence, check you protein concentration after dialysis and make sure there is no precipitation of your protein. You can also add some reducing agent to the reservoir buffer e.g. 5mM DTT if you protein has surface cysteines and there is a risk of aggregation due to disulphide bonds. You can alternatively use 50mM Tris buffer pH 7.5 if 50mM Phosphate is not proving stable for your protein. I am saying this because some proteins are not stable in 50mM Phosphate and they do crash out of solution. (I recommend you use 50 mM Tris pH7.5 if you are flash freezing and storing your samples in -80 degrees C) Sorry for this long explanation. Pardon me if am explaining things as if you are an undergraduate student. Hope this helps!

Wow, thanks for all the responses!

@Tomas, question: for the pierce spin columns, are you implying to add salt-free buffer to the concentrated sample and re-spin it in the same concentrator tube? I hadn't thought to do that, but I suppose that could work.

@Prof Zoran & Ranjan, I use BCA to check for protein retention, not to determine the actual protein concentration. I've got 300mM imidazole in my sample so it I expect it would definitely disappear. I expect the same is true for the A280 readings.

I don't have much in terms of specialized equipment for protein purification; my lab is primarily genetics based, and I've taken my project in the protein direction (so no, no access to FPLC; I do have access to a peristaltic pump, but it's quite old and doesn't go very fast - maybe 10x the speed of gravity filtration)

@Nuanmat, unfortunately, my protein doesn't seem to precipitate in acetone (or TCA). When I tried this on my sample prior to isolating my protein (as a control), I concentrated majority of the proteins, but not my proteins of interest (as detected by Coomassie staining an Western blot).

@Andaleeb, I haven't tried chloroform - thanks for the suggestion!

A number of comments have mentioned Sephadex beads. Can I use this for a family of proteins of varying sizes? I assumed that 40kD-250kD was too large a size range to use Sephadex beads.

@Mario & Sothinathan, are the Amicon ultra centrifugal/Centricon tubes any different from the Pierce concentrators I linked above? I tried finding differences between them a couple months ago, but decided they seemed about the same...I'm new to protein purification though....

@Renu, do you know if the Zebra columns (Pierce) are any different from the Bio-Rad BioSpin p6 tubes? I happen to have just gotten a sample of the latter that I'm going to try soon, and to my untrained eye, they look the same.

@Sunny, my proteins have a range of pI, all in the basic range (8/9-12/13, based on the predicted pI as well as initial isoelectric focusing...although these IEFs haven't been optimal since the current kept fluctuating from the high resistance, which I think is due to the high salt - wicking the samples helped a little, but not much). That link is actually the dialysis tubing I was using (3.5 MWCO), and was having huge protein loss (upwards of 50% overnight at 4*C, and I was using the special clips, not tying the tubing, with a spinning bar at the base of the buffer). My predicted protein sequence lacks cysteines, so I don't think reducing agents would help keep my proteins "happy" in solution. As for replacing phosphate with Tris, are you suggesting this replacement for the affinity column, or for during dialysis, or both? I had considered replacing the phosphate buffer with Tris, but the tech rep from ClonTech (where my nickel resin is from) suggested against it, but didn't give a substantial reason.

Again, thank you for all the responses!

Hi,

all the spin columns are more less the same, only the design differs (which may be sometimes quite annoying (own experience)). It's completely perfect to add fresh buffer, I'm doing that almost on daily basis during my purifications ;)

As of the Sephadex, the important thing is the resolution of your beads. You can have beads for resolution of proteins say 80 - 300 kDa, but beads with resolution of small molecules like G25 or G50 are suitable for desalting, because all the proteins elute in one peak in the beginning.

You have a large MW protein (40-250kDa) you could used a dialysis membrane of 14 KDa cutoff to get ride all salt, you would need extensive dialysis against Milli Q water under gentile stirring or you could use PD-10 column for desalting.

u can use dialysis columnn...

I suggest you Amicon centrifugal filter. It is simple and cheap.

I really can not understand why many of you give the same suggestion. First at all all organic solvents at acidic conditions can easily precipitate proteins from buffered solutions, but not so easily at so high imidazole concentration. Next centrifugal desalting device is not so good as gravity desalting column. And the best one is GE Sephadex G25. I have HPLC, but I do all desalting at small PD 10, or home made packed in small 10 mL syringe. It is good for all proteins with MW higher than 8 kDa

For SDS PAGE and WB you can simply precipitate your proteins with 10 % TCA (final). And finally you can freeze dry your sample after salt removal.

Because not everyone has FPLC/HPLC and even buying the spin columns is probably cheaper than the chromatography column.

Buying the spin column is certainly cheaper, but disposable syringe is much cheaper, easily scalable from 0,5 to 50 mL and such "column" can easily be regenerated.

I think you can use a cheap an fast method like spin columns or Penefsky columns, It´s easy and fast but need some material like insulin syringes and sephadex. You must fill the syringes with sephadex and equilibrate with 10 times your interest buffer, semi dry by centrifugation (around 1000 rpm) 1 or 2 mins. Then you can load yor sample but this time fit a microtube at the end of the syringe in order to get your desalted sample. It´s posible that you miss some protein but not a lot. Easy, cheap and very effective.

Thanks for all the tips! Concentrating my sample in a Pierce desalting concentrator (15ml down to under 200ul) followed by using the BioSpin desalting columns seems to have done the trick!

@Miroslava, for the syringe method you described, do you just assemble the PD10 in the syringe like a gravity column?

Yes. Small piece of some plastic sinter or cotton plug, and Sephadex G 25. But your combination is just fine.

If your protein is easy to purify - which it sounds like it is due to its inherent NiAg binding properties, why don't you next time just make your imidazole elution buffer with low salt. Or do a step-wise wash with decreasing salt concentrations - then elute in low salt

You can use G-25 resin and do chromatography to elute proteins and salts in different fractions. this will take just 15-20 min. Be sure that the protein sample volume is one third of your resin volume in column. To get sharp peaks in elution just increase the flow rate.

In my experience the best way to desalt is using a membrane dialysis in water or buffer low or without salt. Remember the cut-off and change liquid 3 or 4 times per day.

I realize this is an old thread, but---our group has just published this protocol for continuous diafiltration - - basically it's a spin column  with a large reservoir on top (Amicon Pro) so that, depending on your starting sample volume, you can achieve very efficient desalting in many fewer spins than the traditional spin columns listed above.  Check out the paper:

http://www.ncbi.nlm.nih.gov/pubmed/24364216