Hi,

I'm definitely not an expert, but could your observation have to do with potential different strengths of interaction between the various partners? That you lose some of the partners that exhibit a weaker interaction in the current experimental setup?

Hope this helps.

Hi Eef,

Thanks for your answer and that is very well possible.

Hi 

I am also doing similar work and when it comes to interpreting data, what I do is to compare my data with already known interacting proteins and levels changes in case of interaction.

This would just help you to be confident of your data. For me, interpreting novel interaction and their corresponding level changes is tricky thing to comment on. 

Do you expect your protein of interest to promote Autophagy or Ubiquitin Proteasome System mediated degradation? 

I don't know how much my suggestion would help you, but surely it would help to be confident pertaining to your data

Best wishe 

Sorry but I cannot understand what you exactly do when you say "forward experiment".

What forward experiment means? By the way have you "cleaned" using an irrelevant antibody or just beads before doing the "specific" immunoprecipitation?

Hi Rafael,

I used DML-Mass spec (chemical labeling), and forward experiment means Heavy to light ratio where my POI pulldown is heavily labelled and control was light. in the reverse experiment, this labeling is just reversed so if 1 protein is enriched 1 fold in 'forward`' experiment, in the reverse it would be -1 or de-enriched in similar fashion. 

Hi Joel,

Thanks for your answer. Unfortunately, I am characterizing this protein or rather one certain domain of this protein and not much is known about its other interaction partners.

I wanted to know whether theoretically/conceptually you can say from an IP-MS data that certain protein levels were down regulated or not. 

Hi Tanmoyita,

From your reply to Raefel and me, I understand that you want to understand basically level changes of interacting partners of your particular domain of interest.

Did I get you rightly?

If yes, Are you checking it with different conditions or in a same given condition, you are labeling your proteins to see enrichment of interacting partners?

Because, My another doubt here is, How will you get your interacting partner in your light labeled Control expt; ideally you should not get. and if you are getting because of interacting partner's bead binding tendency. you can look for may be fold enrichment in your Test and Control.

However, I am not sure whether such results are acceptable or not.