We plan to use BODIPY™ TMR C5-Maleimide to detect free thiols in our protein samples and have designed two approaches. We would appreciate any advice on which method might be more suitable.
1. Plate reader method: After staining the protein samples, we load them into a 96-well plate and measure fluorescence (Abs544nm, Em570nm) using a plate reader. A blank is included for each condition, and the true fluorescence value is calculated by subtracting the blank from the sample.
2. SDS-PAGE method: After staining, we run the protein samples on SDS-PAGE, followed by fluorescence imaging to visualize the protein bands. Samples without free thiol should not show color after staining.
Since our lab has not performed this type of assay before, which method would be more effective for detecting free thiols? Additionally, are there any tips for optimizing the experiment to ensure reliable and accurate results?
Thank you for your insights!
In method 1, do you have a separation step to remove unincorporated (excess) dye? If the fluorescence intensity of the dye is about the same whether or not it reacts with the protein, there will be no detection unless you remove unincorporated dye.
Method 2 provides the separation method, namely SDS-PAGE. This method will definitely work. The unincorporated dye will probably run at the front. If the dye concentration is too high, it may streak through the lane, in which case you can soak the gel in methanol-acetic acid destain, with a few changes of the destain solution, to remove it and decrease the background.
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