The Dot Blot

Bhuvi M -

Hello everyone! This week, my primary focus has been summarizing my Trimester 1 research for presentation at SARSEF. Today, I am eager to delve into a pivotal aspect of my study—the dot blot analysis, an intricate method that unveils crucial insights into the activation status of receptor tyrosine kinases (RTKs).

A dot blot can be thought of as a molecular detective technique designed to identify specific proteins in a small cell sample. It operates by placing a minute amount of the sample on a specialized paper and utilizing antibodies—akin to precision-trained agents—that selectively bind to the proteins of interest. Unwanted elements are then washed away. Subsequently, a secondary agent enhances the visibility of the targeted proteins, analogous to shining a spotlight on them. The intensity of this spotlight correlates with the abundance of the respective proteins in the sample. While the dot blot provides a rapid overview of protein presence, it lacks the precision of more quantitative methods.

To unravel the RTK landscape in GD versus OE33, we opted for the R&D Systems Human Phospho-RTK Array kit. The selection of this kit stems from its proven efficacy in revealing activated RTKs associated with aggressive cancers. Although not a pinpoint quantitative method, this approach provides qualitative data, illustrating the presence of activated Axl and c-Met in GD and suggesting nuanced differences. Additionally, our observations unveil a downregulation of the EGFR family in GD compared to OE33, confirming GD’s resistance to afatinib, an EGFR inhibitor.

The activation of Axl and c-Met, the latter labeled as its ligand HGF-R in the accompanying figure, suggests potential variations in mesenchymal markers. This finding is particularly noteworthy as it identifies Axl and c-Met as promising therapeutic targets, especially in the context of afatinib resistance.

Additionally, it’s essential to recognize that the dot blot analysis marks only the initiation of our exploration. In the upcoming weeks, I will discuss how I validated these results using RT-qPCR.

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    Hi Bhuvi, I love your description of how the dot blot works. What might be some more exact, quantitative methods? Are there downsides to using these methods?
    Alana Rothschild
    Hi Bhuvi! How did your presentation go??? Thank you for such a detailed post with pictures. I can't wait to learn more over the next few weeks. Good job!
    Hi Bhuvi! I remember the dot blot analysis from reading your research paper earlier this year! Excellent work :)
    Hello Ms. Bennett, the qPCR method is a quantitative method that is commonly used. However, it is more time-consuming because each gene needs to be tested with separate samples. Another important thing to note is that this method targets the mRNA encoded for the receptor instead of the protein. This can be a downside because the mRNA may encode for the receptor but there is a chance that this gene isn't activated during regulated cellular processes.
    The presentation went well! I learned some techniques for experimentation methods that I haven't been able to perform successfully such as culturing organoids, which was very helpful!

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