Hey all,
I am interested to study a transcription factor that usually present in cytoplasm; however; upon activation of certain signaling pathway its transfer to Nucleus.
I want to study that which cells stage the signaling pathway is activated and then the transcription factor localized in the nucleus. To do so, i want to established a flow cytometry protocol where i can permeabilized the plasma membrane only (not nuclear). So it will enable me to calculate the total content of that the transcription factor in the cells and will subtract the cytoplasmic content.
I search online, but I didn't found any reliable paper with data on it. There is a one article published by Beckman Coulter (10.1002/cyto.a.23103). However, they didn't disclose the kind of buffer they used, and mention to contact the company for purchase. I also reached the company, but didn't hear back from them, i think they may stop selling those buffer.
Anyone who have experience in quantifying cytoplasmic versus nuclear localization in the same cell using flow cytometry ?
Thank you so much!
Waqas Nawaz Quantifying cytoplasmic versus nuclear localization of proteins within the same cell using flow cytometry typically involves a process known as intracellular staining and flow cytometry analysis. Here are the general steps to perform this quantification:
Materials Needed:
- Cells of interest
- Antibodies against your protein of interest (one for total protein and one specific to the nuclear fraction)
- Permeabilization buffer
- Flow cytometer
Procedure:
It's essential to optimize each step of the protocol for your specific experimental conditions, including the choice of antibodies, fixation, and permeabilization buffers. Additionally, controls and validation experiments should be included to ensure the accuracy of your quantification.
While the Beckman Coulter article you mentioned may not have provided the buffer details, you can try reaching out to other researchers in the field or your institution's core facilities for guidance on suitable permeabilization buffers and protocols. Collaboration with experts in flow cytometry can also be valuable for developing a robust assay.
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