I am aware that not adding a reducing agent to Laemmli buffer before elution can reduce the amount of antibody contamination in your samples. However, I would have thought that DTT should not be added at any stage of the experiment because it might damage the antibodies? Nonetheless, I have found protocols that both do add DTT and do not.
Hi, usually I don't add DTT into my lysis buffer, although DTT helps reducing sample and helps to exposure the antigen. Instead, if I need DTT to further expose the antigen, I wound like to dilute the concentration of DTT in my buffer before incubation with antibodies and beads (just keep DTT in low concentration to prevent antibodies from damage).
1mM is probably a little high. If your lysate is very concentrated, you can dilute the lysate by lysis buffer without DTT, and the DTT concentration will be lower.
For instance, if I have 5000ug protein in 500ul lysis buffer, I will add DTT free lysis buffer to dilute my lysate to 1000ul . Now the concentration of DTT is 0.5mM, will be better for antibodies to capture the targets.
The best condition for me is lyse cells without DTT and SDS, but use 0.5% of NP-40 if I want to capture cytosolic proteins.
Hope this information can help you!
Diluted it in lysis buffer with DTT woops. hahaha It's ok. I'll see what comes up on the Western and alter the protocol accordingly. Thank you for your feedback.
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