I am attempting to replicate a Co-IP someone else did. They had a defined positive and negative control. My positive control pulls down my protein of interest, but my negative control does as well! My negative control has all of the possible interaction sites for my protein of interest deleted and this negative control has been published to not interact with my protein of interest, yet in my hands it does! The only difference between my protocol and the published protocol is that I do my pre-elution wash steps with the RIPA buffer that I performed the lysis in, whereas in the published protocol the person uses 1X TBS for their washes. Is there a difference between my lysis buffer and 1X TBS that would turn my negative control into a positive one?
I agree that data replication can be infuriating. Regarding your buffer question, if anything, I would say that the RIPA buffer you are using should decrease the non-specific binding relative to TBS, due to the presence of detergents in RIPA. So I doubt it is causing your troubles. Blocking the beads as suggested by Neil is crucial. For troubleshooting purposes you can include some additional controls to determine the pulldown of the negative control, such as beads + protein of interest (don't include the negative or positive controls you mentioned) to see if it truly is binding non-specifically to the beads.
Hi Charles, it can be very frustrating trying to replicate someone elses data. Sometimes it just cant be done. Not knowing what you are trying to do makes it a little difficult (are they membrane proteins, are you using native antibodies or tags, what are you using as the precipitant). If you are using beads to pull down your complex, you should pre-incubate your beads in a BSA solution. Even agarose or sepharose or magnetic beads all have lots of nice protein binding sites. Incubate your "beads" in 0.1% BSA in PBS (or TBS) for 1 hr at 4'C, then pellet your beads and use the pretreated beads to pull down. This should at least get you started.
I agree that data replication can be infuriating. Regarding your buffer question, if anything, I would say that the RIPA buffer you are using should decrease the non-specific binding relative to TBS, due to the presence of detergents in RIPA. So I doubt it is causing your troubles. Blocking the beads as suggested by Neil is crucial. For troubleshooting purposes you can include some additional controls to determine the pulldown of the negative control, such as beads + protein of interest (don't include the negative or positive controls you mentioned) to see if it truly is binding non-specifically to the beads.
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