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Topic: Removing DTT from proteins  (Read 8920 times)

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Offline Corribus

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Removing DTT from proteins
« on: July 16, 2014, 02:47:01 PM »
What is the best way to remove DTT from a protein suspension after a cleavage of a dissulfide bond is complete? I've read that dialysis or chromatography are good options - does anyone out there have a protocol for this? I really need to get rid of as much DTT as possible, with high consistency, since downstream analyses are sensitive to this reagent.

Aside from DTT, are there any other common reagents that can easily cleave a (solvent accessible) disulfide bond in a protein?

What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Offline Yggdrasil

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Re: Removing DTT from proteins
« Reply #1 on: July 16, 2014, 03:55:47 PM »
Dialysis or chromatography would both work.  My personal preference would to use a desalting column as it is a fairly quick method (dialysis may take a few hours).  It will, however, slightly dilute your protein.  If dilution is a concern, you could try using a few rounds of diluting your sample in non-reducing buffer and re-concentrating in a centrifugal concentrator unit.

Another option would be to use TCEP, which is a phosphine-based reducing agent that may interfere less with some downstream analysis than thiol-based reducing agents.  You can even buy bead-immobilized TCEP that you can use to reduce then wash your protein off to recover your reduced protein without any trace of reducing agent.

Offline Corribus

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Re: Removing DTT from proteins
« Reply #2 on: July 16, 2014, 04:05:02 PM »
Thanks - I was not aware of any bead-immobilized reducing agents.

Do you know off hand of any references that have a written procedure for any of these options?
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Offline Yggdrasil

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Re: Removing DTT from proteins
« Reply #3 on: July 16, 2014, 04:19:50 PM »
I would start with the manufacturer's instructions from wherever you buy the columns/reagents and modify them for the particular protein you're working with.

Offline Babcock_Hall

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Re: Removing DTT from proteins
« Reply #4 on: July 23, 2014, 08:54:11 PM »
Another option for rapid buffer exchange is a Penefsky (spin) column.  They tend to be less complete than a standard gel filtration column, but they give less dilution.

Offline Corribus

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Re: Removing DTT from proteins
« Reply #5 on: July 23, 2014, 09:40:14 PM »
Thanks for the suggestion. Dilution is less of an issue for us than residual DTT, which impacts the fluorescence yield of quantum dots. Mercaptoethanol seems to work better, but not perfect. I've ordered some of the TCEP Yggdrasil suggested. If it works, I owe him a beer. :P

What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Offline Babcock_Hall

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Re: Removing DTT from proteins
« Reply #6 on: July 24, 2014, 09:29:36 AM »
Prepacked Sephadex G25 desalting columns are sold commercially under the name PD-10.  Prepacked Biogel P6 (polyacrylamide) are also available commercially.  The manufacturer's instructions for both of these columns suggest diluting the sample more than I think is desirable.  Otherwise both have worked well in our hands for removing DTT or 2-mercaptoethanol. The best reducing agent may also depend on the pH of the experiment.

Offline Yggdrasil

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Re: Removing DTT from proteins
« Reply #7 on: July 24, 2014, 03:24:38 PM »
Thanks for the suggestion. Dilution is less of an issue for us than residual DTT, which impacts the fluorescence yield of quantum dots. Mercaptoethanol seems to work better, but not perfect. I've ordered some of the TCEP Yggdrasil suggested. If it works, I owe him a beer. :P

Well, now that I am invested in the result, definitely let me know how it goes :)

I routinely use TCEP to reduce cysteines in my protein prior to conjugation to maleimide dyes.  I will typically have my protein at ~ 1 mg/mL in buffer, make up a fresh stock of 0.5M TCEP, add the TCEP to the protein solution to a final concentration of ~ 1mM, then incubate for ~ 2 hrs at room temperature.  1mM TCEP may be a bit on the low side for the reduction (somewhere around 5-50mM might be better if you do not expect it to interfere with downstream applications).  Degassing your buffers (to remove oxygen) and including 5-20mM EDTA (to chelate metal ions) will aid in making the reduction more efficient (preventing these substances from oxidizing the TCEP before it can react with the cysteines).

Offline Babcock_Hall

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Re: Removing DTT from proteins
« Reply #8 on: July 24, 2014, 07:07:00 PM »
Yggdrasil,

Do you remove the TCEP prior to labeling with maleimide-based reagents.  I have read somewhat conflicting things about this question.

I have recently become a fan of TCEP, even though I have often used 2-mercaptoethanol and dithiothreitol in the past.  However, TCEP is not particularly stable in phosphate buffer.  I am not sure of the reason.  https://www.piercenet.com/instructions/2160647.pdf

Offline Yggdrasil

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Re: Removing DTT from proteins
« Reply #9 on: July 24, 2014, 08:15:49 PM »
I typically do not remove the TCEP prior to labeling with maleimide reagents.  However, TCEP will interfere with the reaction by forming a covalent adduct with maleimides (http://onlinelibrary.wiley.com/doi/10.1002/elps.200305478/abstract), so it is important to have an excess of dye over TCEP.  Typically, I will use a 10:1 molar ratio of TCEP:protein and a 2.5:1 ratio of dye:TCEP in my labeling reactions and get fairly quantitative labeling over 3 hrs at room temp (typically ~70-90% labeling).

However, if you are concerned about TCEP interfering with the labeling, you can always use bead-immobilized TCEP, and remove it prior to reacting with your maleimide reagent.

Offline Babcock_Hall

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Re: Removing DTT from proteins
« Reply #10 on: July 30, 2014, 12:45:46 PM »
Corribus,

If you need to know whether or not DTT or other thiols have been removed, I recommend (and have often used) the Ellman test.  A paper in Methods in Enzymology by Riddles and coworkers was very helpful to my understanding and practice of this assay.

Offline Corribus

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Re: Removing DTT from proteins
« Reply #11 on: July 30, 2014, 01:46:56 PM »
Thanks, I will look into that. Do you have exact reference?
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Offline Babcock_Hall

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Re: Removing DTT from proteins
« Reply #12 on: July 30, 2014, 05:37:36 PM »
Methods in Enzymology, Volume 91, 1983, Pages 49–60.  Reassessment of Ellman's reagent, Peter W. Riddles, Robert L. Blakeley, Burt Zerner

Please let us know how it goes.

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