Frequently Asked Questions
1. What is YFP?
Yellow Fluorescent Protein (YFP) is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. Its excitation peak is 514nm and its emission peak is 527nm. Like green fluorescent protein (GFP), it is a useful tool in cell and molecular biology, YFP is usually explored using fluorescence microscopy. We introduced an enhanced yellow fluorescent protein (eYFP or Venus) into the cells.
2. What is the parental cell line that we use for our clones?
We use the H1299 non-small lung carcinoma cell line.
3. What are the growing conditions optimal for our clones?
Our incubators are at 8% CO2. Our media is RPMI from GIBCO catalog number 21875, and it is supplemented with L-Glutamine.
4. How do we infect the cells?
We have produced an infection with the Murine Leukemia Virus (MLV) that contains a plasmid with the fluorofore YFP. The plasmid via the virus integrates inside the genome and is then translated from mRNA to a protein with the fluorofore inside.
5. Our tagging procedure.
We used two rounds of CD tagging with the red fluorescent protein mCherry to obtain a cell clone with red fluorescence in the cytoplasm and stronger red fluorescence in the nucleus. Custom software used the red fluorescence pattern to automatically distinguish the cell from its background and to differentiate the nucleus from the cytoplasm. The algorithms in the software can also automatically detect morphological correlates of cell states (e.g., cell death and mitosis). We then used this clone (H1299-cherry) as a basis for our tagged protein library. We introduced an enhanced yellow fluorescent protein (eYFP or Venus) into the red-tagged cells by an additional round of CD tagging, expanded the yellow-tagged cells into clones, and identified the yellow-tagged proteins.
6. Which drug we used for the study?
Camptothecin (C9911 Sigma), was dissolved in DMSO (Dimethyl sulphoxide hybri-max), (SIGMA, D2650) to achieve a stock solution of 10mM. In each experiment, the drug was diluted to 10microM in a transparent growth medium (RPMI, 1% PenStrep, 10%FCS, w/o riboflavin, w/o phenol red, Biological Industries, Kibbutz Beit Haemek, Israel). Growth medium (2ml) was replaced by the diluted drug (2ml) under the microscope.
7. Automated time-lapse microscopy movies and protein dynamics.
Cells were grown in 12-well optical plates in an automated fluorescence microscope with autofocus and control of temperature, CO2, and humidity. Each well contained cells tagged for a different protein. After 24 hours of growth, the drug CPT was added, and cells were tracked for another 48 hours. Images in phase, red and yellow were taken every 20 min at four positions in each well. The resulting time-lapse movies had over 200 consecutive frames per protein studied, where each frame contained 10 to 40 different cells. Movies were stored and analyzed automatically, resulting in traces of protein level or protein dynamics, and location in each cell over time.
8. What does it mean: "Picture Fluor" and "Picture Phase" on the entry page?
"Picture Fluor" is a fluorescence image of the tagged-protein, "Picture Phase" is a phase image; both are obtained by the time-lapse microsopy.
9. What is protein dynamics?The protein dynamics in the database represents a quantitative trace of the tagged protein fluorescence levels in the nucleus and cytoplasm produced by image analysis of the movies over time.
10. How alignment of protein dynamics is produced?Alignment of protein dynamics is produced by placing each profile onto a common time scale (hours, following drug addition) with zero corresponding to the drug-addition time point for the corresponding clone. All fluorescence levels are normalized to the total fluorescence at time=0.