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Topic: Which buffer will be suitable for NHS-ester coupling if I need a pH 5.5-6.5  (Read 4800 times)

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Offline Biotech78

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Hi
I know the optimal pH for NHS-ester (activated by EDC/NHS) crosslinking with amines is around 8. But I have a specific need where I need to conduct this experiment at slightly acidic pH.
I also need MgCl2 and NaCl in the buffer. In other words, I am looking for a PBS pH below 7.

Offline Borek

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I am looking for a PBS pH below 7.

PBS is a poor choice for these pH. Not to mention the fact Mg2+ is a sure way to interfere with phosphates and to precipitate.

Perhaps look for some other buffer? Citrate?
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Offline Babcock_Hall

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I suggest looking into Good's buffers, such as MES, which has a pKa near 6.  Many of these buffers are non-nucleophilic (MES is a tertiary amine), which means that they won't compete with the intended nucleophile in a coupling reaction.

Offline Biotech78

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MES with 150mM NaCl and 1mM MgCl2 is what I think will be most suitable in this case. I can test pH 5.5 to 6.5
« Last Edit: February 14, 2024, 03:51:01 PM by Biotech78 »

Offline Biotech78

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Appears that I don't have MES readily available. What could be other options? I need a pH range 5.5-6.5, want to add NaCl (100-150mM) and 1mM MgCl2. I know NHS ester crosslinking is most efficient around pH 8 but I have a specific need where I have to conduct experiment at slightly acidic pH

Offline Babcock_Hall

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https://www.interchim.fr/ft/0/062000.pdf
PIPES and HOMOPIPES are slightly higher and slightly lower in pKa than MES, respectively.  One might make a mixture of the two.  Bis-Tris is  a tertiary amine with a pKa near 6.5.  Citrate has two drawbacks.  It may chelate magnesium, and it might not be resistant to microbial growth.

Offline Biotech78

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I think acetate maybe a good option. Can work fairly well at pH 6 and below. Also apparently no effect on MgCl2 (at least not any that I know of)

Offline Borek

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Above 5.75 acetate will be a rather poor choice, so it hardly convers your range.

Seems like citrate, maleate, cacodylate, bis-tris are all in your range.
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Offline Babcock_Hall

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Cacodylate is a good choice, but it is a little toxic, so don't eat it, LOL.
EDT
Cacodylate can react with dithiols, such as reduced lipoic acid.
« Last Edit: February 16, 2024, 01:49:09 PM by Babcock_Hall »

Offline Biotech78

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Above 5.75 acetate will be a rather poor choice, so it hardly convers your range.

Seems like citrate, maleate, cacodylate, bis-tris are all in your range.

Tris is not a good choice as it contains primary amines which compete with the coupling reaction. I have been doing coupling reaction previously in Acetate pH 4.5. I think I can test it at 5.5 and 5, it doesn't seem to have any other effects I want to avoid (damaging the folding of aptamers). It is only below 4 that some DNA bases are protonated so 4.5 will be too close to the critical pH I want to avoid in the lower range.

Offline Borek

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Re: Which buffer will be suitable for NHS-ester coupling if I need a pH 5.5-6.5
« Reply #10 on: February 16, 2024, 11:27:01 AM »
Tris is not a good choice as it contains primary amines

Not sure what you mean, it is a tertiary amine.

But then I just listed common buffers that fit the pH range, it is quite possible they won't work for other reasons.
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Offline Babcock_Hall

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Re: Which buffer will be suitable for NHS-ester coupling if I need a pH 5.5-6.5
« Reply #11 on: February 16, 2024, 01:48:10 PM »
Tris is a primary amine, but BisTris is a tertiary amine.  I once used tris to block electrophilic sites that were left over after I joined a protein to a solid support; therefore, its nucleophilicity sometimes comes in handy.  BisTris titrated with acetic acid might be a good choice.

Athelstan

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Acetate Buffer: Acetate buffer, composed of acetic acid and sodium acetate, is effective in the pH range of 3.6-5.6. However, it might not be the best choice if you need to maintain pH closer to 6.5.
MES Buffer: MES (2-(N-morpholino)ethanesulfonic acid) buffer is commonly used in the pH range of 5.5-6.7. It provides good buffering capacity and stability within this range.
PIPES Buffer: PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffer works well in the pH range of 6.1-7.5. It's less commonly used but can be effective if your reaction conditions allow.
Citrate Buffer: Citrate buffer can be used in the pH range of 3.0-6.2, with its pKa values at around 3.1, 4.8, and 6.4. It's not ideal for maintaining pH close to 6.5, but it can be adjusted with care.
« Last Edit: April 19, 2024, 03:07:06 AM by Borek »

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