I'm honestly extremely pissed about this paper. Why would you make a GFP fusion to your target, and *not* check that it was localized correctly before you do an inhibitor screen? I mean look at this hot garbage.pic.twitter.com/NWpjNlxcfA
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I'm honestly extremely pissed about this paper. Why would you make a GFP fusion to your target, and *not* check that it was localized correctly before you do an inhibitor screen? I mean look at this hot garbage.pic.twitter.com/NWpjNlxcfA
Just literally *look at your cells* under a fluorescent microscope, and you'll realize that your GFP-Kras construct is all over the cytoplasm where it doesn't belong.
It's not like *many* labs (including mine) have not successfully expressed GFP-Kras and had it localize to the plasma membrane. But I mean, surely they did the control to show that their GFP-Kras construct had signaling activity? (As ours and everyone else's does.) NOPE!
How could this happen? How could their construct be so unlucky when everyone else's works? I mean, they were so thoughtful about tagging the N-terminus of Kras with GFP, since the C-terminal CAAX motif is essential for PM localization.pic.twitter.com/fWeJboFwSs
But wait, what's this? They weren't *just* expressing GFP-Kras, they also had a P2A-mCherry on the C-terminus to serve as an internal fluorescence control. So clever! Could never backfire!pic.twitter.com/RnBoCooYan
Well that's fine, GFP-Kras-P2A-mCherry will produce two polypeptides, GFP-Kras and mCherry. But, uh, what happens to the P2A peptide? According to this Wikipedia figure, it just disappears! And why read beyond Wikipedia when you're planning an experiment? https://en.wikipedia.org/wiki/2A_self-cleaving_peptides …pic.twitter.com/cwaZAseQOe
I mean, you might read @Addgene blog on multicistronic vectors, which tells us: "The 'cleavage' occurs between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a few additional residues added to the end."https://blog.addgene.org/plasmids-101-multicistronic-vectors …
Ah, so GFP-Kras-P2A-mCherry will make pretty much pristine mCherry, but the Kras half will have an extra *18 amino acids* stuck right at the C-terminus of the critical CAAX box. Which is almost guaranteed to disrupt CAAX function, therefore no plasma membrane localization. Oops.
Ending up this inaugural edition of #BootyJournalClub, this paper is a friggin' consolation prize in a Cell Press journal. Hopefully my next edition won't be so ragey.
So, we should expect #BootyJournalClub to be weekly on Thursdays at 12 pm EST? Yes? Will you be sending out the booty article beforehand, or should we wing it? 
Ha! I actually have a longer #BootyJournalClub installment in preparation, on a paper I really liked (also about Kras!), so I may be back even sooner. But once a week on Thursdays could be a goal for the New Year...
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