Please consider the note in the Thermo protocol, which is really critical:

Note: Immediately react sulfhydryl created by Traut's Reagent, because over time the sulfhydryl may recyclize or oxidize to a non-reactive disulfide.

Thank You Michael. After one hour incubation and further passing through desalting column I tested for -SH groups through Ellmans reagent wihtout any delay. But I did not give any presence of -SH groups

Nearly all thiolation reactions are somehow tricky, particulary due to the oxidation tendency of thiols. Perhaps you want to send me your exact experimental report for a brief check? [email protected]

Below you find an article, which discusses some of the pitfalls of Trauts reagent.

http://pubs.acs.org/doi/abs/10.1021/bc00003a007

What happens when you react with excess Ellman's directly after the labeling step, before you begin with the cleanup? (and then run it through the PD10 in order to see if you get fluorescence in your antibody fraction)

In addition, you could bubble all solutions / solvents with nitrogen or argon and work in a reducing / oxygen free atmosphere (glovebox).

What are you planning to do with the introduced SH groups?

hi, when we add truants reagent and then pass it from a nap10 filter, my antibody concentration is lost. can any one help me with the most accurate way to ellute the antibody?

Hi, I am facing the same problem. I take a sample before passing the mAb through the desalting column and I have thiol signals. I take a sample after passing the mAb through the desalting column and have no thiol signal whatsoever. How did you overcome this situation?

@Sneha & @Yesenia: What buffers do you use?

25 mM HEPES, 140 mM NaCl, pH 7.4

I think that Traut's Reagent is not very reproducible. Alternatives, such as SATA or SATP might be preferable.

https://www.covachem.com/sata_n-succinimidyl_s-acetylthioacetate.html

https://www.thermofisher.com/order/catalog/product/26102

http://www.interchim.fr/ft/8/84235A.pdf

i am using PBS

Looks like your proteins are multimerizing on the column by oxidation and then getting stuck. A potential remedy might be degassing your buffers and flushing them with argon or nitrogen.

To test this hypothesis, try with sth cheap and abundant, e.g. BSA, and with and without a reducing agent like DTT, TCEP or lots of BME. If it should work, try and optimize the degassing procedure and then treat your real samples.

Michael G. Weller can we use dealting spin coloums like zeba spin instead of NAP-10 or PD-10. It says the loss of protein is much less.

https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/benchmarking-experiments-zeba-spin-desalting-columns.html

All sephadex-like stuff inside, I don't give much on such claims, as it a lot depends on your protein. You may reduce unspecific binding by blocking with BSA (5mg/ml or so) and/or a nonionic detergent like Tween 20 (0.1% m/v) in the buffer of your desire.

@ Sneha Ghosh: Yes, zeba spin columns seem to be a suitable option. The result will be dependent on the amount, the concentration and the type of your protein.

Michael G. Weller conc is very less around 150ug and maximum 1000ug. It is an immunoglobulin.

Hi Gangula

It is better to use DTT for mAb reduction, followed by assessment of thiols using Ellman's reagent. the following article can help you:

Article Evaluation of Factors Influencing Antibody Reduction for Dev...