What is the best plasmid & protocol for live cell imaging? I have never done this before?

Dear Kristen,

You can refer to Addgene for some simple advice. Here are some useful links that might interest you.

https://www.addgene.org/collections/empty-backbones/

https://blog.addgene.org/plasmids-for-endogenous-gene-tagging-in-human-cells

https://www.addgene.org/fluorescent-proteins/

If you want to co-express both your proteins of interest, you will need to clone them with two distinguishable fluorescence epitopes, such as GFP and RFP. If you want to study their molecule interaction dynamics, you may need to use some fluorescence epitopes, which can be used for FRET analysis, such as mVenus and mTurquoise2.

Good luck.

Cheers.

Noor Rajeeb

To track two proteins of interest using live cell imaging, there are several plasmid and protocol options you can consider. Here's a suggestion:

Plasmids:Fluorescent protein tags: Use plasmids that express your proteins of interest fused with different fluorescent protein tags. For example, you can use green fluorescent protein (GFP) for one protein and red fluorescent protein (RFP) for the other. This allows you to visualize the proteins separately using different fluorescence channels. Tandem dimeric fluorescent proteins (tdFPs): Another option is to use plasmids that express tdFPs, such as mCherry2, TagRFP-T, or mEos2. These proteins self-assemble into higher-order complexes, resulting in enhanced brightness and reduced photobleaching compared to single fluorescent protein tags.

Imaging Techniques:Confocal microscopy: Confocal microscopy provides high-resolution, three-dimensional imaging. It allows you to capture detailed images of protein localization and dynamics within living cells. Time-lapse microscopy: Employ time-lapse imaging to monitor the dynamics of the two proteins over time. This technique can capture changes in protein localization, movement, and interactions within live cells. Super-resolution microscopy: If you require higher spatial resolution, consider using super-resolution techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM). These methods can provide enhanced imaging of subcellular structures and protein distributions.

Protocol considerations:Cell type and transfection: Choose a transfection method suitable for your specific cell type, such as lipid-based transfection reagents, electroporation, or viral transduction. Ensure efficient transfection to achieve robust expression of the tagged proteins. Fluorescent protein maturation: Take into account the maturation time required for the fluorescent proteins you are using. Some proteins need time to mature and reach their optimal fluorescence. Imaging conditions: Optimize imaging parameters such as exposure time, laser power, and acquisition frequency to minimize phototoxicity and photobleaching while maximizing the signal-to-noise ratio. Live cell imaging chamber: Use a specialized live cell imaging chamber to maintain the cells under physiological conditions, including temperature, humidity, and CO2 levels. This helps ensure cell viability during extended imaging sessions.

Remember that the choice of plasmids and protocols may depend on your specific research goals, the characteristics of the proteins of interest, and the imaging equipment available in your laboratory. It's advisable to consult relevant literature, established protocols, or seek guidance from experienced researchers to tailor the plasmid and protocol choices to your specific experimental needs.

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