There are several possibilities to consider to interpret your interesting observations. To be able to consider these possibilities, we need to know the experimental conditions, including whether phosphatase inhibitors (or any other inhibitors) were included in lysis buffer, that resulted in lack of detection of protein of interest. Additionally, the amino acid sequence of the antigen that was used to raise the antibodies. The simplest explanation without knowing exact experimental conditions is that epitope within the protein of interest is not accessible/available to antibody under the experimental conditions tried so far. More thoughts after learning the experimental conditions.

It looks like your protein of interest is regulated by some ubiquitin proteins in the whole cell lysate and some how the recognition of your protein by ubiquitinases is blocked by your MYC tag. Try to lyse the cells with protinase inhibtor cocktail. ANother possibility is the endogenous levle of your protein is very low. try different concentration of protein I would recommend more protein. Good luck

Please tell whether your recombinant protein was not at all visible in gel or it was not detected in immunoblot. If your protein was not visible in gel, that indicates  protein degradation and you can use a protease inhibitor cocktail during preparation of cell lysate.

Hello,

Thank you so much for your suggestions. Sorry if I didn´t give you all the details, but I did not want the question to be too long...

Meanwhile I have stripped the membrane and hybridized again with new antibodies: anti-T7 Tag (to detect the recombinant protein) and anti-MYC (to detect the overexpressed protein in the ox-plant extracts). The recombinant protein is detected with similar signal in both loaded samples: b) recombinant protein and c) a mixture of recombinant protein + plant protein extract. Also, the Myc-tagged protein in the overexpressor plants extracts is also detected! 

Murugesan V S Rajaram, with these results it seems that the ubiquitin regulation does not seem to be responsible for the dificulties. The endogenous level of the protein is also something that I obviously have to consider. For that reason I have included the overexpressor plants extracts, which although not detected by the specific antibodies, were clearly identified with anti-MYC.

Divaker Choubey, your suggestion "the epitope within the protein of interest is not accessible/available to antibody under the experimental conditions" was my major suspicion. In fact there is the possibility of phosphorylation within those peptides, against which the antibodies were raised. I did not know this at the moment... But do you think it is reasonable or possible that all, or the majority, of the protein being in the phosphorylation state? Or adding the denatured recombinant protein to the extract (on ice), that all of it adopts this phosphorylation? Phosphorylation is what came in mind, I can´t remember more possibilities, cause I don´t know much about this...

I have run along in the same gel, dephosphorylated exracts of WT and OX-plants (although I am not sure I used enough alkaline phosphatase) and untreated samples. No detection either.

Also I have run all samples in duplicate in two gels and treated one membrane, before imunodetection with a denaturing solution, described for other cases.

As for the extractions preformed I have tried extracting simply with Laemli buffer, with TCA/acetone or another lysis buffer with Tris 50mM, NaCl 150mM, DTT 1 mM, proteases inhibitor cocktail and MgCl2 10mM. I never got signal in the extracts with any of these. Although with the last one was the only time I tried to add recombinant protein to the extracts, to check.

Is there another possibility you can think of, another PTM or conformation problem that was not addressed in these experiments?

Thank you so much for your help!

Cecilia

More clear now. However, two more questions remain: Do you cut the gel and probe the relevant area of the membrane or an entire membrane?  If the whole membrane, do you detect signal higher than the predicted size of the protein?

I probed the entire membrane and the only signal I get is in the predicted size of the protein

Based on the information available from your observations, it is conceivable that protein modifications of some sort and/or protein aggregation (which makes the protein insoluble, thus, it is eliminated during the procedure prior to loading on the gel) are contributing to your difficulties in detecting protein of interest. 

Thank you very much

Hi Maria,

I also have a couple of questions,  1) your recombinant protein, is it the full length protein or a peptide, and was it made in e coli?  Proteins made in E coli are not the same as native protein which can be extensively covalently modified by gycosylation, phosphorylation etc.  These modifications however unless they are in the Ab recognition site shouldn't stop binding and there fore detection, but rather a shift upwards in the molecular weight. 2) I would suggest that you try an old fashioned dot blot to check if its the reduction of the protein that is the problem in the western.  Take nitrocellulose membrane (not PVDF) and spot your recombinant protein and your plant protein samples on it.  10ul a time and let them dry before adding a second 10ul. Dry the membrane and then wet with TBST buffer and proceed with western blotting.  You don't say whether you are running your protein in reducing or non reducing conditions, this can effect WB.

Hello Caroline Elizabeth Dunk. Thanks for your interest and sugestions and sorry for delayed response...

1) the recombinant protein is the full lenght of protein and was produced in e coli

2)The proteins were run in reducing conditions.

Thanks

Hi Maria, I had another thought do you know if your protein is a nuclear protein,  in which case I would recommend a high salt buffer for the extraction.

Caroline

Hi Caroline. I don´t know, but there is no indication it should be nuclear... Thanks anyway. Maria