I'm using amber suppressor tRNA in E.coli to perform in vivo photo-cross-linking experiments to study certain protein-protein interactions at the outer membrane. I do get indications of photo-crosslinks by running SDS-PAGE, followed by western blotting, but they were very faint bands. Any suggestions to get more of these crosslink products on a gel? Perhaps an enhancement?
Please provide details of your experiment: wavelength (365?), device (power), time, culture density, media
Do you have working positive control that is known to work with amber tRNA?
Hi Tomasz,
I'm using 50 ml LB cultures (grown at 37 degrees) and UV irradiating (365 nm) them for 10 mins (Room Temp.). Also I do have a positive control in-place!
and what is density (OD600) of culture and what device(Stratalinker?) is used to irradiate?
Dense culture and some amino acids (trp) in media may quench UV. Culture with OD ~ 0.5 should be o.k. Keep also thin layer of culture when irradiating.
Does positive control works well in your hands or problem exist for both samples?
I'm using a UV lamp and mid-log phase cultures, irradiation takes place in a Petri dish.
Hi Dilshan,
I am still not sure how powerful is your lamp, but things that are worth to try:
- dilute your culture and/or split into a few Petri dishes (double check does dish is UV transparent if lamp UV irradiate from the bottom). Use media without UV quenchers
- use more powerful UV source
- extend irradiation time (least favorite idea)
If all above fail, maybe you can redesign your amber tRNA construct?
Hi Tomasz,
Thanks for your comments, I better tweek my experimental design a bit then.
Cheers
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