Hi all,

I am currently working on an experiment to determine the optimal concentration of iodoacetamide (IAA) for blocking cysteine (Cys) thiol groups in protein samples for our redox proteomic. I am using BODIPY™ TMR C5-Maleimide as a fluorescent dye to label unblocked thiols, and theoretically, higher concentrations of IAA should reduce the fluorescence signal by blocking more thiol groups.

However, I observed something unexpected: samples treated with very high concentrations of IAA (100-200 mM) still produce fluorescence signals. According to my understanding, increasing IAA concentration should decrease the fluorescence signal. At a certain concentration, the fluorescence value should be close to that of the blank group (lysis buffer + dye + water). However, I still observe relatively high values.

Details of my protocol:

The RFU of the blank group is approximately 300, but the RFU at high IAA concentrations (100-200 mM) are all around 3000. From this value, I can infer that the free thiols should have been completely blocked once the IAA concentration reached 100 mM.

My questions are:

Any insights or suggestions for improving my protocol would be greatly appreciated!

Thank you for your time.