Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Topic started by: Biotech78 on November 10, 2023, 01:43:44 PM
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Hi
Anyone knows the recommended conditions for storage of ethanolamine-hcl solution. I need to use it in an SPR experiment to inactivate the remaining NHS-esters after EDC/NHS coupling step.
However, I have observed that ethanolamine-hcl may not be working properly. It does not cause any (net) RU shift. I suspect I am not handling the solution properly. Plus my analytes are binding to control channels. Dissociation is virtually impossible for both test and control channels indicating proteins (analytes) are binding to the unreacted activated COOH groups on the sensor surface
How do I store the solution? Refrigerated? Frozen? or need to wrap parafilm around falcon/bottle cap? protection from light? what am I doing wrong?
So far what I know is that in solid form its hygroscopic, the solution shouldn't be. I saw one paper recommended make solution and then store in aliquots on -20. Light sensitivity not reported.
I make solution in a 50mL falcon, adjust ph with NaOH (8.5) and store at 4, no wrapping no light protection
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It should be stable if it is a tight stopper on the flask, just glass is fine.
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Can you explain the term "RU shift?"
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Since this is for an SPR experiment, RU stands for resonance unit. 1RU shift is equal to a critical angle shift of 10-4 deg
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As long as the pH is adjusted correctly, I cannot see a problem. BTW, there is some literature on following NHS coupling reactions: DOI: 10.1039/c5ay00042d. My situation was a little different; I tried to use spectrophotometry, based on the absorbance of the side-product (which as a pKa of 6) with mixed success.
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T. Miron and M. Wilchek, A spectrophotometric assay for soluble and immobilized n-hydroxysuccinimide esters, Anal. Biochem., 1982, 126, 433–435.
I recall obtaining some information from this paper in an attempt to follow a model reaction between an amine and a succinimide ester.
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As long as the pH is adjusted correctly, I cannot see a problem. BTW, there is some literature on following NHS coupling reactions: DOI: 10.1039/c5ay00042d. My situation was a little different; I tried to use spectrophotometry, based on the absorbance of the side-product (which as a pKa of 6) with mixed success.
I think you are right. I was talking to the millipore tech support scientist the other day and he said it could be dissolved CO2 lowering the pH. I checked the pH of stored buffer and for some reason, the pH was rather high (9.2ish) rather than what thought I had adjusted it to (8.5). Don't know it was the pH meter which needed recalibration or what. I will make the buffer again and check.
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Borek is far more knowledgeable about pH than I am. However in my experience the pH 10 buffer standard is not indefinitely stable, perhaps because of carbon dioxide. My preliminary thinking is that a different analytical technique from SPR might be worth pursuing; the HILIC method that I cited above has a detection limit of 1 mg/L: Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC). One problem with spectrophotometry is that many other substances absorb where the anionic form of N-hydroxysuccinimide does. I had been running a reaction in an organic solvent and attempted to extract the chromophore to monitor its progress. I think such an idea has merit, but it needed some fine tuning.
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Borek is far more knowledgeable about pH than I am. However in my experience the pH 10 buffer standard is not indefinitely stable, perhaps because of carbon dioxide. My preliminary thinking is that a different analytical technique from SPR might be worth pursuing; the HILIC method that I cited above has a detection limit of 1 mg/L: Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC). One problem with spectrophotometry is that many other substances absorb where the anionic form of N-hydroxysuccinimide does.
My primary aim is not the quantification of NHS. EDC/NHS coupling and then ethanolamine deactivation of "remaining" activated esters (that did not couple with the ligand) are the steps I am doing to prepare the SPR chip. My target analyte is a protein which will bind to the ligand I had already coupled to the activated carboxyl groups at the SPR chip surface.
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I may not be following. Will the reaction of ethanolamine with an activated ester produce NHS?
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no it will quench any remaining suc-ester
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If the pH of the ethanolamine solution is stable it is ok I think the problem is coming from elswhere
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no it will quench any remaining suc-ester
https://www.sciencedirect.com/topics/medicine-and-dentistry/n-hydroxysuccinimide#:~:text=The%20NHS%20ester%20end%20of,hydrolysis%20rate%20of%20the%20ester.
If ethanolamine reacts with a succinimide-ester, N-hydroxysuccinimide will be a by-product.
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If the pH of the ethanolamine solution is stable it is ok I think the problem is coming from elswhere
I am still not able to figure out where the problem is. pH is okay. I recalibrated the pH meter and the buffer pH was around 8.43 (vs expected 8.5) which is within the expected error range. Could it be that the problem is at EDC/NHS step? Not enough esters to react with ethanolamine?
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no it will quench any remaining suc-ester
https://www.sciencedirect.com/topics/medicine-and-dentistry/n-hydroxysuccinimide#:~:text=The%20NHS%20ester%20end%20of,hydrolysis%20rate%20of%20the%20ester.
If ethanolamine reacts with a succinimide-ester, N-hydroxysuccinimide will be a by-product.
I have a COOH group on my chip surface, I add EDC and NHS which leads to the formation amine reactive NHS esters. I add a ligand, say protein or any other molecule with an amine group, it reacts with esters and forms a stable amide bond. Now not all the esters would react with my ligand so if I bring in another protein, it's amine groups will also react with the remaining esters and form an amide bond. This is what we dont want. We want this last protein (analyte) to bind to the ligand only. So we use any primary amine to "block" or "inactivate" the "surplus" esters. This is where ethanolamine comes in. Ethanolamine will block the unreacted esters so my analyte will not have any esters to react with and will only bind to the ligand.
I am not concerned what are the products of reaction between ethanolamine and esters
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I am not concerned what are the products of reaction between ethanolamine and esters
What I am trying to do is to question (in a constructive way) your choice of method. What evidence from the literature is there that SPR can detect the success of the reaction that blocks the surplus esters? I suggest considering one of two methods found in the literature as alternatives. BTW I coupled alkaline phosphatase to a Sepharose 4B activated as the 6-aminohexanoic acid N-hydroxysuccinimidyl ester long ago. I used Tris to quench, but I did not think about trying to follow the quenching reaction to completion; so I admire your efforts in that regard.
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I am not concerned what are the products of reaction between ethanolamine and esters
What I am trying to do is to question (in a constructive way) your choice of method. What evidence from the literature is there that SPR can detect the success of the reaction that blocks the surplus esters? I suggest considering one of two methods found in the literature as alternatives. BTW I coupled alkaline phosphatase to a Sepharose 4B activated as the 6-aminohexanoic acid N-hydroxysuccinimidyl ester long ago. I used Tris to quench, but I did not think about trying to follow the quenching reaction to completion; so I admire your efforts in that regard.
You may have a point. SPR may not be ideal to detect the success of the reaction we are talking here but it can nonetheless indicate how successful the reaction was. If ethanolamine does react with the esters on SPR chip, there will be an RU shift compared to baseline. If not, all the ethanolamine will be washed away in the wash step. Aim of my experiment is SPR itself. Ester-ethanolamine reaction is just a "preparative" step and because it is for SPR, I have to do in "on chip". All I can say is that is all I can do in the given circumstances.
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Last night I went to the library and into the journal stacks (which may not be available much longer) to re-read the article (Miron and Wilchek 1982 Analytical Biochemistry 126(2):433-435) on using spectrophotometry to follow coupling reactions involving NHS esters. When I consulted this article before, I was running a reaction in an organic solvent and attempting to extract N-hydroxysuccinimide into the aqueous layer of a micro-extraction. One thing that limited the usefulness of this technique in my hands was that there was a large value of the absorbance at time zero. This might have been due to the reagent itself or to an impurity in the starting materials.
I am not trying to beat a dead horse, but if there were some way to perform UV spectrophotometry in your application (I know almost nothing about SPR chips), the spectrophotometric method might have more than one use. One of their applications was to measure how much active ester was found on commercial gel designed for coupling to macromolecules, and I can imagine that this might be useful in your work as well.