Which protein are you referring to? Is the protein intracellular, extracellular?

Assuming that your positive control for the immunohistochemistry is the same tissue/cell type with just different conditions, it is possible to assume that the technique is probably ok.

There are a lot of reasons why mRNA is not translated...

As the poster mentioned above, we will need more details about what you are looking for, how high was the message expression, etc to help.

Barring technical issues, the fun answer is that this gene could be subject to post-transcriptional regulation. If you know an upstream regulator (RNA binding protein, miRNA) you can use a method to get rid of that and see if your protein expression appears (shRNA (crude, but should work), sponge, respectively). Or, maybe the protein is not subject to heavy post-transcriptional regulation and instead you are just not looking in the right place- i.e. some unexpected localization or cell type. Many mRNAs are non-coding, so this is not completely unexpected- here is a nice review- granted it's neuro specific (Article Long Noncoding RNAs: Central to Nervous System Development

Technically speaking, you could try to detect the protein on a Western Blot. If that works, your antibody may just not be good in IHC. You could also try another antibody. Sometimes proteins are easier to detect in ICC vs IHC, so many try that. As a positive control, you could clone the gene under a constitutive promoter and do and an over expression just to see if this gene is ever translated. Maybe the levels are just too low to be resolved by microscopy. Sometimes I really can't tell if a protein is present under 20x but can under 60x magnification. Maybe borrow someone's confocal (if not already using that...)?

Better to use western blot to be sure and from there u can use activity assay if u want to dig in deep

Thank you very much everyone for showing interest. Interestingly, I have done different isoforms of transport proteins in the same tissue those are intracellular. Some are detected well and some are not with the same protocol. The gene expression is lower (1/2 fold) for the not expressed proteins than expressed proteins but are detectable.

If the protein is very low in concentration it is possible that it might not be detected by IHC?

I will plan future experiments but right now trying to understand the different possibilities of what has been done already.

With the example of CKIT that is expressed by many tumours but not necessarily associated with gene mutation ther reasons are listed below. Given the high percentage of immunopositive melanomas (48%) and PAM (50%), the usefulness of CD117 staining as a screening method to identify conjunctival melanomas that would benefit from imatinib therapy (ie those with (ckit gene mutations) is addressed. Besides KIT-activating mutations, additional mechanisms may drive CD117 overexpression. Constitutive phosphorylation of the receptor protein (Antonescu et al. 2007), a decrease in protein turnover resulting in accumulation and thus overexpression of C-kit in malignant cells or stem cell factor ⁄KIT autocrine loop alterations (highlighted in uveal melanoma cells; Lefevre et al. 2004), could contribute to tumorigenic mechanisms also in conjunctival melanoma. In conclusion, KIT-activating mutation appears a rare event in conjunctival melanoma but CD117 has not proved to be a good predictor of its presence. Nevertheless, performing the molecular screening to identify Kit molecular alteration in conjunctival melanoma patients may theoretically allow to offer a tailored treatment in those patients that could benefit from therapy

I realise I have explained the reverse. Also there are missense mutations and nonsense mutations that are not functional or transcribed.

Alka, I think the short answer to your question (can a very lowly expressed protein be undetectable by IHC) is yes. I would caution this by adding that since most IHC is indirect, every staining with a secondary is actually an amplification of signal, so in most cases proteins are over-represented in IHC in terms of how much is there in a cell.

Some clarification here:

"The gene expression is lower (1/2 fold) for the not expressed proteins than expressed proteins but are detectable."

When you say that your gene expression is "1/2 fold lower" for some isoforms compared to others, I imagine this is based off of qRT? It must be since you call these "non-expressed." Are you basing your judgement of "non-expressed" off of not detectable by IHC? This could be because of low levels or it could also be that the antibody is not good in IHC. Again, as others have recommended, I would do a Western blot to see relative protein levels, if you haven't already.

Are you certain your antibody can distinguish amongst isoforms? As I said before, some antibodies just don't work well in IHC or IP as they do in WB. Do you know in what situations/contexts certain isoforms are preferentially translated over others? These are some more questions to think about as you proceed.

Hey Tatiana,

1. Yes "1/2 fold lower" is by qRT

2. When I say protein is not expressed, it is by IHC. "not detectable" is a possibility I am thinking of but as you said IHC can even detects lower protein concentration, so now I will think of other possibilities.

3. I have used antibody of Abcam as I didnt want to take chances with the quality. I have tried two different types of antibodies as well and both worked well for my positive control tissue. However, from both the antibodies I didnt get any stain in my samples.

4. I have not tried WB but i will try in future. But the question still remains for IHC.

5. These isoforms are highly specific to different tissue types. Translation according to me should be there as the background studies indicate that these proteins should be active in the sample tissue but which isoform is active is still a matter speculations.

It could be that you just need to boost your signal. I would recommend increasing the amount of antibody you use (you could try multiple dilutions to see if any work), along with increasing the duration of primary antibody (or incubating at room temp), and possibly increasing secondary by a slight amount.

Even though your positive control worked, it could just be that the signal is just not nearly as powerful. There are several resources out there to help boost your signal.

Since this is tissue, did you try antigen retrieval? Were the animals perfused? These can help increase signal as well.

Good luck!

Hi,

There are a lot of examples in which gene expression is present but protein is not detected. A good example is gene coding HIF 1 alpha protein. The HIFA gene is expressed independent of oxygen condition but protein is detected only in low oxygen tension. In the high oxygen tension environment, protein is degraded. In this process participate HIF prolyl-hydroxylase.

I think you can try different dilutions from your primary antibody and see what you’ll get

best

Thank you everyone for your suggestions. I have tried different dilutions (1:100 to 1:10), different antigen retrievals, time of incubation and different secondary systems as well (different brands and concentrations).

@Justyna: Thank you very much for your answer. I am looking for molecular understanding and it was very helpful.