Generally EDC-sulfo NHS activation is done for carboxylic acid moeties on a nanoparticle surface or a planar substrate.
Can we do EDC-Sulfo NHS activation for small molecules of around 300 daltons which have a single COOH group ?
I have a small molecule with a COOH group. I want to conjugate this to BSA through EDC-SulfoNHS cross-linking chemistry. I tried few times without any success.
Are there any particular experimental procedures for doing this ? Has anyone worked on activating COOH groups on small molecules ? What could be the possible reasons that would prevent this reaction from happening ?
Thank you very much for your time
How do you know that the conjugation has not worked? Another approach would be too use a very high concentration of the small molecule, if you can, and simply add EDC. The activated carboxyls are likely to react with other carboxyls to form anhydrides, which should then react quickly with amines on the BSA. EDC concentration should be significant lower than carboxyl conc. (i.e COOH from the small molecule) so that the EDC is rapidly consumed. The reaction then involve small molecule to BSA conjugation, rather than BSA to BSA. You can always add the BSA a little after the EDC if you have any issues with BSA cross-linking. However, you should not wait more than a minute or two as none of the reactive groups generated have a long half life. A pH around 6 should be okay. Use MES buffer at 50 or 100mM. Also make sure the pH is still okay after you have dissolved your small molecule, readjust to pH 6 as required.
Dear Nick,
Thank you again for your help, for your time and valuable inputs.
I would like to briefly tell you the procedure I followed so that you will know where I went wrong.
Step 1: I took small molecule carboxylic acid (SMCA), EDC and Sulfo NHS in 1:3:3 (ratio in terms of no. of. moles) in phosphate buffer of pH 5. Incubated this mixture for 45 - 60 min at RT @ 850 rpm.
The above step I also tried with Phosphate buffer of pH7.4
Step 2: After 45-60 min of incubation, I added this mixture of SMCA+EDC+SulfoNHS to BSA pre-dissolved in carbonate buffer of pH8.5 and incubated this new mixture overnight for 12 hours. Then I used 30KD centrifugal column to separate the unreacted SMCA (filtrate) from the SMCA-BSA conjugate, if formed (retentate)
I used different ratios (in terms of no. of. moles) of SMCA:BSA like 12:1, 27:1 and 3000:1.
I used a colorimteric chemical test established for this particular SMCA to detect its presence in retentate and filtrate. The test showed that SMCA is present in the filtrate but not in the retentate. Thats how I confirmed that SMCA has not conjugated with the BSA in the retentate.
Also, I spotted this retentate on a nitrocellulose membrane of a lateral flow assay and used Gold NP- Ab of SMCA on the conjugate pad. I ran the strips with PBS. But I did not get any color at the region where I spotted the retentate which is supposed to contain SMCA-BSA. This also confirms that SMCA has not conjugated to the BSA
Hope, I am clear.
Coming to the method you described, looks like there is no need to use sulfo NHS? Also, is it a single step method where in you add SMCA, EDC and BSA almost at the same time (or BSA is added 2-3 min late) ? Is the pH of this single step mixture needs to be at 6 ? How much time does this reaction go ?
If possible, can you let me know the no. of steps involved and their details. Are the molar ratios I used appropriate ?
Thank you again for your time. Really appreciate your help
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