Pavla,

Thanks for the clarification. Couple of suggestions follow.

RT-PCR Is pretty sensitive, so do you know if the immune complex is bringing down a selection of cellular RNA's in a non-specific manner? One test would be to look for other commonly expressed cellular messages in the precipitate, by RT-PCR.

How do you "bond" the RNA to the protein? and then how do you "unbond" it for the gel for the western blot? If SDS doesn't do it the bonded RNA might stop the complex entering the gel or eluting from the gel efficiently.

Hope this helps

Dear Pavla, I am not all that clear on your experimental design. If you could describe it more clearly for me then I might have something more substantial to add. In particular I do not know how the immunoprecipitation fits in your experiment.

I think that you will find that WB can be a substantially less sensitive technique than qPCR for detection of gene expression. All I can recommend at this point is that you test different antibodies and optimize the conditions for detection of your gene of interest by WB (e.g., load more protein, test different primary and secondary antibody dilutions, use a high sensitivity detection assay, etc).

Well, by no doubt summer is not the best time carrying out WB measurements!  Yet, the issue you are dealing with is entirely different.  qPCR indicates that gene activity is expressed through mRNA release. However, that result no necessarily implies that mRNA should be translated into proteins. Indeed, this result deserves a more in depth analysis as it evidences that gene activity is not followed by de novo protein synthesis. Why not!  May be some problems within the splicing machinery of the protein synthesis apparatus is currently working on in your experiments. I suggest you to investigate a) some markers downstream the biochemical pathway you are dealing with in order to substantiate the 'loss-of-function' linked to the undetected WB-protein levels; b) to assess if the gene activity could lead to some mutated proteins (therefore not detected by usual antibody-kit); c) to evaluate the gene-protein network in another cell line to ascertain if such an effect is limited to a specific cell-line.

Best,

Mariano

I'd agree that RNA does not necessarily mean protein, and that the RT-PCR assay will in any case be more sensitive than the Western. I also am unclear exactly what your procedures are, but one trick to look for a poorly translated protein is to transfect 293T cells and look. These cells transiently transfect very efficiently and consequently make a LOT of protein. You can usually see an order of magnitude more protein compared to most other transfection protocols.

Thanks all of you but I didn't explaint it clearly, I'm sorry. The aim of this experiment is not to detect expression of some protein. The main aim is to detect splicing protein bonded on plasmid construct.

So experimental design was at first optimization of immunoprecipitation and to detect splicing protein of interest (naturally occurring in cells) itself by WB - this was OK. Second step was to detect specific RNA from plasmid construct which should be bonded on this protein. In this step I was able to isolate specific RNA from immunoprecipitate but I wasn't able to detect specific protein by WB - but with simple logic, there SHOULD be specific protein after WB when I have specific RNA and non-transfection control is clear (how it should be).

Once more thanks for any suggestions

Pavla

Pavla,

Thanks for the clarification. Couple of suggestions follow.

RT-PCR Is pretty sensitive, so do you know if the immune complex is bringing down a selection of cellular RNA's in a non-specific manner? One test would be to look for other commonly expressed cellular messages in the precipitate, by RT-PCR.

How do you "bond" the RNA to the protein? and then how do you "unbond" it for the gel for the western blot? If SDS doesn't do it the bonded RNA might stop the complex entering the gel or eluting from the gel efficiently.

Hope this helps

As far as I know this protein is binding to many RNAs, it is transcriptional factor. Maybe I will try to find in literature some housekeeping gene with describe bond of this protein.

During the process I "bond" the RNA to the protein by UV-crosslinking but after immunoprecipitation I devide the eluate to the two parts. From first part I isolate RNA (after proteinase K application) and second part I use for WB without any "unbonding". Maybe this is the problem, you may be right. I'll try it once again with "no UV-cross control" and let you know! Thank you once more! 

Pavla, thanks for the clarification.

Assuming that the protein is the limiting factor, or close to it, then it is possible that most of it is "bound" to nucleic acid. As such, you will likely observe band (or more likely several bands or smear given the various nucleic acid species that you protein binds to) that is shifted up on a WB. The suggestion of no UV-cross control is a good one, just make certain that the detergent is sufficiently strong to disturb the protein-nucleic acid interaction. Even if it is not, you may see "naked" protein run at the correct size and protein-nucleic acid complexes run at a higher size.

Best.

I think there must be some experimental error. Redo the experiments by being very vigilent at each steps. Western Blot should give you the results.

Hi everybody,

I'm sorry for late response (lots of work, as usual in laboratory). However the problem of RIP moved forward little bit.

1) Positive RT-PCR after RNA isolation from immunoprecipitate was due to genomic DNA (short DNase treatment).

2) IP from Hela cells still didn't work so I changed them to HepG2 cells - now the IP is working (positive WB). How is it possible, I'm not pretty sure.

3) Unfortunately, now I'm stucking on RNA isolation. I'm trying several approaches (RNA isolation kits, phenol-chlorophorm extraction) with no result. I treat the immunoprecipitate by preinase K before RNA isolation (inc.30min 55 C) and now I want to add RNase inhibitor step. I hope it will be  succesfull.

Do you have any experience with RNA isolation from immunoprecipitate?

Thanks again! 

Pavla

I think you have answered the mail your self.