Jul
31

Post #7: So many colors!

Hi everyone! This past week, I have been working on my co-transfection experiments with mutant tc-TRa1 and aggresome markers, GFP-170 and GFP-250. After not observing any colocalization with the autophagy marker, I didn’t know what to expect in this new round of experiments. I suspected I might see some colocalization here and there, but that overall, my results would look somewhat similar to what I saw with LC3.

I could not have been more far off. The red and the green came together to give me a beautiful yellow color!

I am still working on obtaining high-resolution images that clearly show the colocalization of tc-TRa1 with the aggresome markers. Moreover, I am hoping to quantify the colocalization of these proteins by using the Pearson’s product-moment correlation coefficient. However, to do so, I need to change my microscopy techniques. I am currently working with a fluorescence microscope, which is awesome for obtaining images of GFP-tagged proteins inside the cell and giving me an idea of whether proteins are being localized to the nucleus or the cytoplasm. However, its power does not come close to the power of a confocal microscope. 

Put simply, confocal microscopes use the power of a high intensity laser and a spatial pinhole to create a crisp, high-quality three dimensional image of the sample. This means that instead of simply looking at my cells in two-dimensions, I would be able to move through my sample horizontally and vertically and observe if my protein is actually in the nucleus, or if it’s on top or below another cellular structure. Moreover, I would be able to calculate Pearson’s correlation coefficient!

Our confocal microscope is currently undergoing maintenance, so unfortunately, I might not be able to obtain all of my data before the end of the summer session. However, I am excited to start working on this as soon as the fall semester rolls around! Over the next few final weeks of the summer, I will continue reviewing the literature on autophagy and aggresome formation. I hope to find some information that helps me understand and see the connections in my preliminary results. Stay tuned!

 

 

Comments

  1. clopezsilva says:

    Hi pwvolante! Thank you for your feedback. I’m sure your shades of brown soils and minced up leaves look just as beautiful as my cells!

    Colocalization is the observation of spatial overlap between two different fluorescently-labeled samples. For example, in some cells, I might see my GFP (green) protein just in one part of the cell, and my mCherry (red) protein in a completely different part of the cell. This is not colocalization. However, in some cells I might see the green and red protein located in the exact same location in the cell; they are overlapping each other! Hence, the yellow color we observe. This is colocalization 🙂

  2. pwvolante says:

    Wow, this sounds a lot more pretty than the samples I get to look at, which are all various shades of brown soils and minced up leaves. Could you explain what colocalization is? Sounds cool!

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