How to analyse the TNBS assay?

Hi guys, 

I'm currently working on the modification of Lys residues and need a quantitative assay to proof the content of free NH2 afterwards.

Is there a good protocol with TNBSA (2,4,6-trinitrobenzene sulfonic acid)? 

Or do you use another agent? Because some literature says TNBSA is highly specific for primary amino groups (Lys + N-terminus), but other paper tell you that it will also react with e.g. Cys... Does anybody know?

Thanks for your help =)

Ina

Hi everybody!

I would like to quantify the hydrazide functions in a sample by TNBS assay, and I am wondering why do we need to use SDS? 

Second question, why do we need to work at pH 8? is it in relation to the hydrazide pKa?

Thanks in advance for your answer

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Kindly help me.

Estemeed colleagues,

I found some issues regarding the quantification of the data for TNBS assay.

There are different protocols on how to perform that but it is clear to me the "fil rouge" that connect all of them.

However, I don't get why in many articles the authors do not show the formulas used to calculate the moles of amino groups per gram of gelatin starting from the absorbances they recorded during the experimental part.

Do the formulas change depending on the protocol you are following? Or is there a universal formula for all the protocols?

Can anyone help me to solve this issue, please?

Kind regards

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