Was trying to figure out exactly why the BME turns the column brown, found this post; "I've had this same problem and contacted GE Healthcare's tech support and it was an easy solution. The brown color comes from the reaction of the reducing agent with the "free nickel" that is bound to the column. Pre-wash the column with high mM imidazole elution buffer first and then equilibrate with binding buffer. If you do this, the column won't turn brown." So the BME and DTT are reacting with the "un-coordinated" free nickle on the column, and a 0.5 M immidazole buffer will wash it off. Just make sure to get rid of all the immidazole before loading your sample.

Hi Keya,

My comments are specific to the below His-tag resins, since there are many types of His-tag resins available and they have different properties. I use 5 mM BME in the lysis/sample buffer for Talon resin (Clontech) and it works fine during the 1 hr batch incubation (that it the max recommended concentration/time for BME), I do not use BME in the wash and elution buffers (though I do add BME to the eluted protein fractions). You can also use BME and DTT up to 10 mM with Roche's Complete His-tag resin, but your lysate has to be at pH 8.0, and the binding is weaker so you have to be careful about adding imidazole to the lysate to prevent non-specific protein binding.

BME (Beta-mercaptoethanol) is more stable than DTT so it is a better choice for the purification of proteins which can take hours at higher temperatures.

What is the name of the His-tag resin you are using and what company are you purchasing it from?

Hello Gary,

Thank you for your answer. I use His trap (5ml) Fast Flow (FF) column from GE healthcare. The booklet that came with it says that the column can withstand upto 5mM DTT, 20mM BME, so on. But I face the Ni reduction problem with 1mM DTT.

Hi Keya,

since 1mM DTT obviously is to high for your Ni column, I would use BME as Gary already suggested. Actually DTT is not recommended for the NiNTA (Qiagen) at all, I guess the GE system works quite similar.

Best Kai

Thanks Kai I was thinking of doing that. Will start with 5mM BME and see what happens.

I have used the GE His trap FF columns in the past with 5 mM BME in the lysis buffer and it would turn the column brownish in color but the color went away when I washed the column with buffer A that did not contain BME. The protein did bind to the column and eluted fine with an immidazole gradient (again with no BME in either buffer A or B-with immidazole).

Was trying to figure out exactly why the BME turns the column brown, found this post; "I've had this same problem and contacted GE Healthcare's tech support and it was an easy solution. The brown color comes from the reaction of the reducing agent with the "free nickel" that is bound to the column. Pre-wash the column with high mM imidazole elution buffer first and then equilibrate with binding buffer. If you do this, the column won't turn brown." So the BME and DTT are reacting with the "un-coordinated" free nickle on the column, and a 0.5 M immidazole buffer will wash it off. Just make sure to get rid of all the immidazole before loading your sample.

Thats a wonderful bit of information. Thank you very much. I will most definitely try it out.

Thank you all for the answers. I tried washing the column first with buffer that had 0.5M Imidazole. This turned the column very blue. I then washed it off and re-equilibrated it. also, I did not put any DTT in the binding or elution buffer, just in the lysis buffer. And it worked..!! I have some protein now. I appreciate all your suggestions.

Thanks for letting us know you got it working.

Great that you've solved your problem, but if you need to have reducing agent around during purification of a his-tag protein, I would suggest using TCEP, at about the same concentration (0.5-2mM). It is more stable and persistent than DTT or bME, doesn't smell (it is not volatile), and it won't turn your column brown.

Thanks Oliver will look into it (never liked the DTT/BME odor and associated toxicity), also thinking a 0.5 M imidazole washing step is good too for the His-trap media since it will get rid of all the free nickle which otherwise would bind to the His-tag and then wash off the column before elution. But with Talon resin, I think this is not an issue, since I have not seen discoloration with 5 mM BME (no imidazole pre-wash).

Hi All, I had similar nature of issue with objective of IB solubilisation and about to work between DTT or BME as choice. What would be ideal conc range to work for borh.

had you tried native or denaturing conditions of Hist purifications after reducing agents treatment. Kindly share your inputs. In one method it is shown that DTT can be dialysed agaisnt the IGEPAL at 0.2 % Tris pH 8.0 before purification. Looking forward to your valuable suggestions and comments.

Roche sells a resin that is compatible with 5 mM BME (maybe DTT too) called complete his tag resin.

Takara/Clontech sells a His-Tag Protein Miniprep column that is supposed to be compatible with BME up to 30mM and DTT up to 10mM.  Might be worth a shot.  You can request a sample, and they will send you two microcentrifuge columns. 

http://www.clontech.com/US/Products/Protein_Expression_and_Purification/His-Tagged_Protein_Purification/ibcGetAttachment.jsp?cItemId=103486&fileId=6960763&sitex=10020:22372:US 

Use TCEP. It doesn't reduce nickel columns. It's an all around better reducing agent than BME or DTT. It's more stable and less pH sensitive. It doesn't smell bad. We rarely use reducing agents other than TCEP with proteins.

@Ho Leung Ng. 

Can you brief the exact concentration range of TCEP to be used for all the buffers in Ni-NTA chromatography. I am already using 0.5mM TCEP but my protein is still aggregating. Can I use it in size exclusion buffer also?  

0.5 mM TCEP is reasonable. You can use TCEP in the size exclusion buffer too. In some cases, the thiol in BME/DTT can reduce disulfide bonds that TCEP does not. I have heard colleagues use up to 10 mM TCEP in difficult cases.

I had already tried 0.5mM TCEP but still my protein is aggregating, can i use 1mM TCEP. Is it too high? 

You can try much higher than 1 mM TCEP. Something else to try is iodoacetamide to alkylate cysteine. But your protein can be aggregating due to factors unrelated to cysteine crosslinking.

Hello,

How did you get the brown color off the ni column? 

Hello,

You can also use TECP ! It doesn't reduce the column ! but a bit expensive as compared to DtT, BME.  hope it helps.

For removing brown precipitates, I tried once to use 1% APS (the one for making polyacrylamide gels, mild oxidant) for at least 10 min; set the flow rate at 1 ml/min. Still working but I wouldn't take risk anymore, 'cause that freaked me out!

Hi all,

I have accidentally added too much TCEP in bacteria extract and it reaches about 15 mM. Do you think I can load this on a Ni column?

Thank you

Hello,

I had a similar issue recently with a protein that required a minimum of 5mM DTT, otherwise it would precipitate in solution. Rather than make new buffers, or try more expensive reducing agents, I simply used the same buffers with no DTT, but I eluted my sample into a test tube that had DTT in it.

I.E. I eluted 20mLs using normal buffer into a container that had 20mLs of 10mM DTT.

This resolved my issue. Although, I have noticed 5mM BME does not cause severe issues with the column. While I have noticed damage over time and the yellowing/browning you see, it is 100x less severe than using DTT.

Hi all,

the INDIGO Ni Agarose from our company, basing on a new chelator, is stable against at least 20 mM DTT and EDTA, without losing nickel and binding capacity.

It even works better with DTT, don't ask me why...

https://cube-biotech.com/products/purification-resins/his-affinity-resins/indigo-ni-agarose-100/368/purecube-100-indigo-ni-agarose

Best, Roland

Try elution with Imidazole

I struggled with this issue before. I use 5 mM DTT and 1 mM EDTA (stabilizes DTT) for my purification (both are the maximum amounts the GE manual recommends using). I found that the column turned brown immediately when using this buffer.

I solved the issue by cleaning the column after recharging with 0.1 NiCl. I wash with 2-3 column volumes of binding buffer (the recipe is on page 8 in the attached document) then with 2-3 column volumes of elution buffer (to get rid of excess nickel).