I have been conducting a series of experiments where I transiently transfect plasmid with my gene of interest into HT29 cells. When I performed qRT-PCR on these cell samples, I can clearly see an over expression of mRNA, which is significantly higher than the initial null colon cancer cells. However, in western blots, I cannot detect the protein at all. What are the reasons that mRNA transcripts are not translated into protein? Also, are there any other techniques that I can use to detect the protein expression without it being too strenuous (like Mass Spec)? I have considered conformational changes in the protein because of which the antibody may not be recognizing the epitope. Are there any ways for me to detect it using the same antibody as the commercial antibodies available for my protein of interest are very minimal and so far this seems to be the best one? I have used the same antibody on a different cell line and have been able to easily detect endogenous levels of my protein of interest without any problems.
Hi Vidya,
Alternate strategy perhaps?
One quick and easy method to detect if your protein of interest is being expressed is to perform an Indirect immune fluorescence microscopy. this will hop you visualize the localization of the protein within the cell as well. Additionally you may need to semiquantitate the signal intensities and compare to those of your controls, (Empty vector transfected) to show overexpresssion.
I have encountered problems as such, but it has typically been related to issues with antibodies, for example, same cat# but a different lot no, an older aliquot etc. For this reason i usually try to test and optimize for more than one antibodies (when possible). But I have always found that semi quantitative microscopy helps with respect to determining how many cells in the population have actually taken up the plasmid and are actually expressing the gene of interest.
If you need help with any of the protocols please feel free to email me.
All the best ;)
First question: Are your antibodies working? Then it could be targeted degradation of the protein by the cell. Its also possible, that splicing etc. is not correct, so the protein is degraded.
Does your gene have introns? In that case, if you cloned your gene from genomic DNA instead of cDNA, those introns could be a problem if the HT29 cells aren't able to splice them.
There are various technical issues and mechanistic possibilities (as directed by Dr. Sajesh and Dr. Praetorius, and 3' and/or 5' UTR mediated regulation of translation or proteosome mediated degradation etc; https://www.researchgate.net/publication/257348973_Blood_pressure_homeostasis_is_maintained_by_a_P311-TGF-_axis) as long as your gene of interest (cDNA) is in frame. I know one of my friends having similar problem because of vector (commercial). He had successful expression of his protein after switching the plasmid. Polyclonal antibodies are advantageous for your studies, if available. Other possibility, If transfection efficiency is low, you will see PCR/qPCR amplifications with undetectable levels of protein. More controls in each step will troubleshoot your problem faster.
Article Blood pressure homeostasis is maintained by a P311-TGF-β axis
I would also add that if you have abundant expression of mRNA off of plasmid it can actually overwhelm the cell's translational machinery and you can end up with far less protein. This may be overcome by using inducible promoter rather than the standard SV40 in most plasmids.
If your antibody and PCR primers target different parts of the protein/mRNA then you may be measuring different things due to alternative splicing and nonsense-mediated decay. Plus, mRNA levels don't always correlate with protein levels as each molecule has its own turnover rate. It could be that the protein turnover rate is very different between your cell lines.
Finally, why is mass spec. not an option? You've already got the gels, so just cut the band out where your protein should be and see.
Thank you so much for all your suggestions.
Dr Badri, I am very certain that the insert is in the correct frame. I will try and order a new plasmid and try again to see if protein expression increases. I am currently using a polyclonal antibody. I have not checked the transfection efficiency of the HT29 cells.
Dr. Sajesh, I would very much appreciate a protocol for indirect immune fluorescence microscopy. I will use an empty vector as my control for the experiment to quantitate my findings.
Dr. Praetorius, I am very sure my antibodies work as I have used a positive control cell line that expresses my protein of interest in abundance for my western blots. Is there any way to stop targeted degradation of the protein by the cell? Maybe add in a proteosome inhibitor (such as MG132) to my cell lysates?
Dr WInter, I have cloned the gene from cDNA and not genomic DNA.
Dr. Beazley, I have performed a gradient experiment, where I transfected the HT29 cells with increasing concentrations of the plasmid. So, even at the lowest concentration of plasmid that shows a good mRNA transcript level and no subsequent protein expression.
Dr. Cole, I will check which are the target regions for both my primers and antibody and see if they are different. I was hoping to do a quick test as opposed to Mass Spec to check if I could try and solve the problem. However, if it does not work, the next thing would be to definitely MS the bands cut out from the gels.
How about a qPCr assay targeting different regions of the ORF,and a 2D gel elctrophoresis along with a control (control plasmid) for differential expression analysis?Was there a reference used during qPCR assay and then a comparison between transfected non-transfected cells?
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