I am planning to do Co-IP. My lysis buffer has 1mM DTT. If I use lysate with DTT Can this affect my immunoprecipitation experiment ?
Can I store immunoprecipitated beads in 4 C for ON till I complete my western ?
Since the chains of immunoglobulins are held together by disulfide bonds, which are reduced by DTT, it is possible that DTT would interfere with the immunoprecipitation.
You can definitely give it a try. Dilute your IP lysates with IP buffer without DTT. In my experience upto 0.5 mM DTT have no effect on co-IP experiments.
You can store your beads for ON after washing.
It could through what Adam B Shapiro mentioned, but another thing to keep in mind is the DTT could affect the resin that's used. DTT is a no-go for TALON resins but ok for others, sepharose, FLAG, etc.
In my experience, I used 0.5 mM DTT in my co-IP experiments and found that it has no effect in this concentration.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials Chemistry
- Polymer Chemistry