Hi Ahmed, 

Things similar to your case happened to me before. Sometimes experiments just don't work without any reason, although theoretically it should work. 

To your case, I would suggest you check if your protein exists in the flow through. If yes, you can use the flow through for further purification, because although your protein does not bind to the column, other proteins including GST have been captured in the column. You may concentrate the flow through, check the purity, and a further purification step using gel filtration may help you get the protein purified in a satisfied level.

Hi there,

Just a few lines of thinking:

Have you tried on column digest by thrombin in order to selectively release your protein of interest from GSH-agarose?

What pH range are you working at for ion exchange chromatography?

Have you tried strong ion exchangers in a first move?

Hi Ahmed, 

Things similar to your case happened to me before. Sometimes experiments just don't work without any reason, although theoretically it should work. 

To your case, I would suggest you check if your protein exists in the flow through. If yes, you can use the flow through for further purification, because although your protein does not bind to the column, other proteins including GST have been captured in the column. You may concentrate the flow through, check the purity, and a further purification step using gel filtration may help you get the protein purified in a satisfied level.

Hi Ahmed, the purpose of most protein affinity tags is to do a single step purification procedure without the need for additional purification steps.

Do you have problems with thrombin or other proteins that are being copurified?

@Dominique, je suis aussi Charlie.

@ Tarik, please don't copy/paste things from Wikipedia without acknowledgements, it is very bad form.

 http://en.wikipedia.org/wiki/Ion_chromatography

Perhaps you could try an ammonium sulfate precipitation followed by gel filtration or hydrophobic interaction chromatography.

Sometimes a protein will not adsorb to ion-exchange resins because the protein is highly aggregated. You can check for this by running the protein on a gel filtration column, which also can potentially help in the purification.

Thank you so much Mr.Nasser for your kind offer

Dear Ligar, Yes I did it, but I got really low yield. Therefore, I decided to purify my protein and then digest it with thrombin. I used different PH ranges . For example,  I used PH 8-9 with the anion exchanger and PH 4.5-5.5 with cation exchanger. Yes, I used strong ion exchangers ex. HiTarp Q ff and Resource s 1ml. 

Hi sun,

It seems my protein concentration is not much in flow through, but the GST tag concentration is really good. by the way, both proteins ( GST tag  and my protein have almost the same conc. after cleavage and before Ion exchange). Is there more suggestion.

Thanks 

Hello Stefansson,

I wont my protein in high purity and do not have issue with thrombin. Thank you for helping though.

Thank you so much Mr. Nesbitt and Mr. Shapiro. Are there any recommendations or suggestions to how avoid the aggregation in case my protein has this issue?

Regards,

Hi,

it's not clear if (and if yes, why) you try the ion exchange step right after the cleavage reaction with thrombin or if you run a second time over the GSH column in order to separate the cleaved protein from the GST and uncleaved fusion protein which should be the first thing to do after cleavage. Using ion exchange directly makes things more complicated because you have three (in best case) proteins which you have to separate (GST, uncleaved GST-fusion and your protein, I don't coun't thrombin for the moment but that's the fourth one although in low amounts). Depending on their properties it might be not so easy to find conditions where all of these are well separated, that's why affinity tags should be used first and as much as possible.

If your protein just "disappears" after the column, it seems likely that it forms aggregates which could indicate that it forms soluble aggregates with GST right from the beginning.

Another often underestimated reason for "disappearing" protein can be be old or "dirty" columns which were not cleaned regularly / properly when they are re-used so protein and other stuff just accumulates on the beads and leads to more protein aggregation especially with sensitive proteins. We use 6M guanidinium every time and NaOH from time to time to keep the columns as clean as possible.

Aggregation can sometimes be reduced during expression by slowing down expression. One way to do this is to lower the temperature of induction. Another approach that may be used is to coexpress a chaperone.

Aggregation of purified protein or protein during purification may be reduced by changing the buffer conditions, such as by using higher or lower salt concentration, changing the pH, including a mild detergent, or adding a stabilizer such as glycerol, sucrose, or trehalose. Keeping the protein concentration relatively low could also be helpful. If you know of a ligand for your protein that you can obtain inexpensively, try including it during extraction and purification to stabilize the protein.

Tarik, is impossible for you to copy/paste information without acknowledging where it is from:

http://www.labome.com/method/Protein-Purification.html

As far as I can understand from your different postings you applied your digested sample to cationexchange at pH 4.5-5.5 or to anionexchange at pH 8-9. You saw mainly GST in the flowthrough, but could not elute the target protein by NaCl? That would suggest that your protein is still on the column. Did you try elution by pH change (increase for cation exchange, decrease for anion)? Do you see a peak when CIP'ing the column? As others have suggested you should run the digest through the GST column again before the next step to remove the tag and undigested fusion protein.

@Tarik Nasser: Why are you doing this?!?!?! Copy/pasting such a long text that could just be linked with an URL is just disrespectful both to the original poster (because you're assuming he/she is not smart enough to find this on wikipedia or elsewhere), to the others who have to scroll down your spam posting to read the other answers and the original author whom you are not mentioning! Plus your text is not even complete (figures missing!), so please stop doing this!!

@Ahmed Dhamad: Sorry for this, but if you let people get away with such behaviour this forum could become useless very soon.

Have you checked the MW of your protein and GST tag? If you see they are different, so you can try gel filtration. 

Hi, I totally agree with Steingrimur and Huseyin and he must at least refer the article to the original author. However, in same countries, the researchers do not know much about copy right. 

Hi Bo, I have not tried to elute the protein by changing the PH. Thank you for your suggestions.

Hi U.Nguyen, the M.Wt. of my protein is a little bit different form the GST and I am trying to use IEX right after affinity chromatography to separate my protein from 1- thrombin which has M.Wt similar to my protein 2- other proteins.  

I am really appreciate and thank all researchers who are trying to solve my issue . Now I am trying to apply some your suggestions.