Happy Belated New Year! We've all gotten back from holiday travel and have been busy the past couple weeks carrying out the first steps in our plan. We've grown up cultures of our proinsulin-gene-carrying e. coli, induced them to express it, and are now working on extracting the proinsulin and verifying that we've got it.
I'm writing from our weekly Sunday afternoon work session at CCL. Patrik says: "Today we spun down the cells that we induced to produce protein yesterday. We're sonicating them to break them open, and then we'll purify the proinsulin with a nickel column. Also, the bottle of LB buffer looked a bit cloudy yesterday, so we'll also make and autoclave some more LB broth to be used on Tuesday or Wednesday."
We ran a couple gels with the output from the nickel columns over the past weeks, but haven't gotten clear bands that would tend to confirm that we've got proinsulin.
As Isaac wrote, "The most common, inexpensive way to check to see if you have made a protein is by SDS-PAGE gel. However, the effective range of standard SDS page tends to start around 10 kDa, and insulin at around 6kDa is much smaller than that... In order to reliably detect insulin, I routinely made by hand and ran custom 10-25% PAGE gradient gels." Proinsulin is a bit bigger but we may still be encountering this kind of problem, so we may need to move on to mass spec, or try something else, depending on how our next few attempts go.
Once we get the proinsulin purified and verify that we've got it, we'll be moving on to the really novel cutting and folding work. Look out for updates on this and more on our progress from other members of the team soon!
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