Dear scientific community,
I faced an unexpected problem regarding the detection of a protein in the murine muscle stem cells. I am interested in a GPCR, which has a relatively low mRNA expression (RNA-Seq). Anyway, on a couple of immunostainings I was able to detect it. I assume that the IF worked correctly, also the secondary AB only (Alexa 594) showed no specific signal at all.
To make life easier, and also for live-cell imaging, I ordered a transgenic mice, which has a hrGFP reporter sequence at the end of the last exon of the GPCR of interest (global knock-in). The mouse is published already, and I also detected the hrGFP signal in the positive-control tissue.
But I was quite surprised that the hrGFP signal was not detectable/on the level of autofluorescense in the muscle stem cells, that I am interested in, even though I saw the clear signal with the IF.
Have anyone experienced such situation, when a reporter signal is much lower than the signal of IF? What would you suggest to try?
Thanks in advance!
Best,
Michail
Dear Michail,
Maybe you can try boosting your signal with a GFP-antibody. You said that RNA-levels were quite low, but that you did detect an IF signal. Remember that (indirect) IF always causes signal amplification.
Hope this helps!
Best wishes and good luck!
Dear Michail,
Maybe you can try boosting your signal with a GFP-antibody. You said that RNA-levels were quite low, but that you did detect an IF signal. Remember that (indirect) IF always causes signal amplification.
Hope this helps!
Best wishes and good luck!
I agree with Rebecca--- I have dealt with transgenic animals in the past where expression levels of the reporter GFP was quite low. As she mentions, it is common for simple amplification using IF (GFP antibody) to visualize. You would likely have more nonspecific signal with IF alone (and even with the GFP antibody), than endogenous fluorescence from a TG animal (depending on how strongly expressed the gene of interest is).
