Hi,
I am encountering a problem with protein expression and than detecting on western blot. I have around 120kDa membrane bound protein which is now fuse to GFP(~150kDa) to help in selecting positive cell when I transfect HEK cell. However I am experiencing a high cell mortality with ones that are emitting green signal, when I tried to do western blot I am detecting a band of 120kDa and not ~150kDa which should be the correct size for GFP+Protein of interest.
Therefore its puzzling me whether the GFP is getting cleaved at some point from my protein, is there a way to detect where and what could be the possibilities of GFP getting cleaved.
Utilizing fluorescent proteins as imaging probes is a widespread and multifarious technique in microscopy. GFP-tagged proteins can be acclimated to track and examine genuine-time localization, interactions and translocation of proteins of interest as well as to investigate several aspects of cell biology. The Y-box binding protein 1 (YB-1) is a pleiotropic protein involved in a wide number of cellular processes and a substantial quantity of erudition on YB-1 localization and function was engendered utilizing YB-1-GFP constructs. Recently, it has been shown that YB-1 plays a critical role in cell stress replication, playing a consequential role in the formation of stress granules (SG).To deeply investigate this subject, we engendered a stable HEK293T cell line constitutively expressing YB-1-GFP. In physiological conditions, YB-1-GFP expressing cells deported like the parental cells and YB-1-GFP protein congruously localized to the cytoplasmic compartment. However, upon oxidative insult, we optically canvassed a vigorous abbreviation in cell viability along with the occurrence of unwonted GFP-positive aggregates. Taken together, our findings suggest critically revising subsisting insights obtained with the YB-1-GFP construct and, more in general, to beware and be critical in interpreting data obtained with functionally uncharacterized GFP fusion proteins. https://dailyplanet.club/
- Badges
- Science method