I have found 27 host proteins as interacting partner for the bait protein (Viral protein). out of them I have selected 3, all of them grew blue colonies on QDO?X/A. On colony lift filter assay, distinguioshed blue patches could be seen and relative b-galactosidase units were more than 5 in ONPG assay. for positive control (p53 and SV40T), b-galactosidase units were 20. but I am not able to confirm this by either Co-IP or IF. I am using santacruz antibodies but I am not able to detect the pre host protein in total cell lysate also. Please help. My thesis is stuck
It's entirely possible that you won't be able to confirm a "real" interaction by every method. Yeast two hybrid screens, depending on the expression vectors you're using (and a variety of other factors, including yeast strains and growth conditions), may provide positive results when other screens fail to detect an interaction. Failure to detect a target protein in your lysate seems like a bigger problem - do you need to use whole cell lysate or can you filter by size first?
Do you mean yeast lysates or transfected mammalian cell-lysates?
In addition, what do you mean by ''pre host protein''?
As mentioned above, there are technical differences between approaches. But depending on your Bait, there could be some amount of false-positives in Y2H.
Good Luck
I concur with the previous two answers, but would also add a few more things. Your inability to detect your host protein in whole cell lysate should be your greatest concern. It may be due to a number of technical issues with your blotting technique. I would consider the following:
1. If the total cell lysates are not the yeast extracts, are you sure the host protein is actually expressed in the cell type you are analyzing?
If the answer is yes, then how did other people detect it? You should replicate their methods as much as possible. If you're not sure, a literature search may help you identify a good positive control cell type for you to include. This would also give you a starting point for how to prepare your lysates and probably a good antibody for detection.
2. How many different antibodies to your host protein have you tried in your blotting?
I would try to obtain a small aliquot of a number of different antibodies to your host protein if possible, ensuring that you include both N- and C-terminal-directed Abs. Further, I would obtain them from multiple suppliers. This way you maximize your chances of finding an antibody that works. If this is a reasonably new protein with not much known or few antibodies, this could be difficult!
3. How many ways have you prepared your total cell lysates?
Detection of proteins by blotting is CRITICALLY dependent on your lysis conditions. I typically use at least 3 different lysis buffers +/- sonication when trying to detect a new protein in lysates. I can send you the recipes for the lysis buffers I've used in the past. You could then screen each of these lysates using the different antibodies you obtained in No.2. above!
Hope this helps! Let me know if you want the lysis buffer recipes.
Hello, Thanks everyone.
I am using mammalian cell lysate transfected with the bait protein to detect endogenous host prey protein. Till now I have tested four cell lines (Hela, BHK-21, CHO-K1 and LFBK), two lysis buffer (pierce RIPA and non-denaturing lysis buffer with 0.25% tritonX-100), 2 antibodies (one polyclonal and one monoclonal from SantaCruz) but I am failing to get the target prey protein in Co-IP as well as in whole cell lysate. Though i got images in immunofluorescence and I am attaching the same for confirmation from your side that whether thay are really colocalizing. i am really new in all of these techniques
@Daniel plz provide the lysis buffer recipes any other tips to help.
@Manoj it was typo error I meant host prey protein
Dear Sonalika, If you cannot detect the proteins by western, I will be really careful about considering these stainings as specific (even though epitopes behave differently in IF versus SDS-PAGE).
As others have mention here, your main issue is that you cannot detect the prey proteins by western (interaction / colocalization study will be secondary). I see that you have tried many cell lines and it is hard to beleive that none of these lines express any of your targets. But still you can have a look into database like Human protein atlas for the relative expression levels of these proteins in these cells lines. In addition, if you are dealing with some strange cytoskeletal proteins, it is also possible that you are not solubilizing them enough for detection. So it will be a good idea to try to detect the proteins by western in cell lysate prepared directly boiling in 2x SDS-gel loading buffer. This will solubilize almost everything (even though a little slimy and not good for interaction studies, it will let you know whether you really have the antigen in the lysates).
The second part is whether you are achieving good enrichment with you bait protein. Even if you manage to detect the endogenous band for the prey proteins, if your transfection efficiency is not great, then you will not know what to expect. For example, if you have your overexpressed bait in only 20% cells and input to Co-IP ratio for the endogenous prey will be skewed towards negative side. Sometimes detecting these interactions in co-IP (with endo protein) will require extensive standardization (Y2H can catch some very weak interactions too!!!). It will be a better idea to start with co-expression of both tagged proteins (prey and bait) and pulldown (GST, His, Flag tags) and only going for endogenous with positive prey clones. We normally try to start with the Y2H clone (sometimes truncations), then full length clone and then endogenous if the first 2 are successfull.
Good Luck
Thanks Sir, I have thought about gst pull down but my bait protein-GST tagged is expressing in inclusion body so i am afraid I could not use it for pull down. any suggestions for this. if i used his tagged vector, truncated bait proten could only be expressed.
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