Meet AGATA:

Meet AGATA:

21.10.14, 17h00 Venture Challenge EPFL, Lausanne. Closed.
27.10.14, 17h00 Sictic Investor Day, Lausanne CH. Program.
28.10.14, 18h00 Bioscope inauguration, Geneva CH. Program.

How to get the most from your luciferase assay

Bring the lysate and the luciferin at room temperature before reading!

As low temperatures minimize protein degradation in a cell lysate, working in ice is a classic attitude in molecular biology. This is a good reflex, but sometimes inappropriate. If you directly transfer in a luminometer a 96well plate previously kept on ice, the first wells will be read at ~4°C. But wait, for each well the luminometer takes ~20 seconds (10'' for firefly and 10'' for renilla). Thus, a full plate will take 20s * 96 = 1920 sec = ~30 minutes. The last wells will be likely at room temperature. The temperature bias could sensitively affect your readings!

Want to check? Work on ice, and prepare a big lysate from cells transfected with luciferase and put 20 uL in each well of the 96well plate (alternatively you can use recombinant luciferase diluted in a phosphate buffer containing 1mg/mL BSA). Directly transfer the plate from ice to luminometer and start reading. You will observe something similar to the figure:



Firefly luciferase is an enzyme from an insect. Insects are poor thermo-regulators. Therefore most of their enzymes optimally works at room temperature. This is the case for luciferase. During your reading, the increase in the temperature sustains firefly's activity. As you can see, this phenomena efficiently balances some luciferase degradation. People is usually scared by protein degradation, but in this case it is worst to keep your plate in ice. For your comfort, you can quickly bring a plate at RT just by incubating at 37 °C for 3-4 minutes immediately before reading. Pimp your results!

See also my post on luciferase downsides.

new protein software

PDB VISUALIZATION
I'm not a protein scientist, but sometimes I need to look at a protein structure, for instance to check if a putative interaction motif is exposed on the surface and whatsoever. The problem is that I need to do this just 4-5 times in a year, therefore I never invested my time in perfectioning my script abilities on rasmol for example. It is really a pain to get back to the instruction manual every time. Now, I found this new online software which runs in the cloud without plugins: JOLECULE, maintained by Bosco Ho.
Jolecule is designed to make it easy to find and look at and identify residues and atoms. [...] Just edit the url, add "/pdb/' + PDB code. (For a new structure, the server fetches the PDB from the RCSB)
For instance, here there is the glucocorticoid receptor bound to DNA.
With JOLECULE it is easy to save you preferred view, you can also annotating it and
share your protein annotation on the web with a simple link.

Pretty easy and very intuitive, you just need a PDB code and some patience the first time when you address the server to a new PDB (the server is still a small one). The good news is that once loaded, the PDB is cached on Jolecule server forever.

PROTEIN DOCKING
If you are just curious to simulate the binding of any molecule (written in SMILE) on any PDB, and you don't know nothing about docking (like me) just play with SWISSDOCK (and than, speak with someone on the field).