A few months ago, I finally got around to installing & learning NAMD for doing some molecular dynamics. Installation was (and has been) relatively easy. Leaning how to use it in the past was always a bit too much for me for some reason or another. However, at the encouragement1 of a new postdoc in one of the adjoining labs at work, I gave it another stab with much better results!
Working with a newer fluorescent molecule on the fluorimeter today, I wanted to verify the (old) solutions of it were all the same thing1. Just running a typical emission profile on the same dilution of each to make sure they were similar concentrations of the same compound. Averaging five repeat spectra for each, I noticed the emission intensity was dropping a bit after the first pass, so obviously there is some concern for photobleaching.
Considering some of my planned assays are looking at 5–10 minutes long for the kinetics to reach equilibrium, I wanted to see what I was working with in terms of photobleaching. Perfect time to take lunch! I setup a 1 hour scan—albeit probably overkill—to look at the time frame & extent of photobleaching. Coming back from lunch, I was a bit confused…
Granted, this is all normalized against a stable section of the scan (35–45 minutes in), but I wasn’t expecting that obvious of a signal drift, considering the lamp had been warmed up for an hour before even the multi-pass emission scans before!
I’m slightly concerned because (a) these are within the scope/timeframe of the kinetics that I’m looking at, and (b) this is likely an issue with the excitation source, either the power supplying it, or the bulb itself.
Either way, I’m a little concerned about this. I’m not looking forward to having to examine the full length of time needed for signal stabilization (if it can be stabilized at all, presuming it’s not a power supply/line issue). What I really need is some sort of an internal control I can use, especially considering I have two detectors at my disposal. I was attempting to find out if I can use the RCQC signal to measure variance in this, but in some subsequent scans I saw no correlated signal variation in it with the signal drift. However, the drift I saw may have been poorly characterized kinetics for the proteins involved.
Soooooooooooo, yeahhhhhhhh…fun times. *sighs*
1These came from an older time with other students in the lab, so there are some “hazy” (if not completely absent) details about them.
Some days, you’re just better off not going back to the original experiment in its entirety.
I opted to return to the original buffer system for some tryptophan fluorescence in vitro previously done in the lab. Up until now, I was using a different buffer that I had optimized for doing fluorescence anisotropy back during the fall. Using the cross-optimized buffer, I could see 20-30% reductions in tryptophan fluorescence (after accounting for the 10% reduction in signal from ligand addition to induce the conformational change).
Now? Either I’ve gotten way too slow at returning the reaction to the fluorimeter for analysis (doubtful), my protein is crapping out (also unlikely as this is a pretty fresh stock), or this buffer just doesn’t play as nicely. I’m getting maybe a 5–10% change in signal upon ligand addition. And I’m like 3 hours into this 4.5ish hour set of samples.
As I’ve mentioned previously, some of the command line tricks for batch processing PDB coordinates for electrostatics models throw some errors back at you, although they don’t implicitly prevent you from using APBS. They just make the surface models look a bit ugly, as you’ll have conflicting charges confined in an unnatural environment, yielding bizarre electrostatics representations (I’ll have to generate an image sometime).
Starting my work with radioactivity1 again today, I took it upon myself to try and track down a lab coat that fits. The lab has a small collection of archaic2 lab coats that I looked through, but unfortunately none of them fit. Because they were just all wadded up & shoved into the shelf (and because I’m a bit of a neat freak), I opted to fold them back up to save some space. As I was working through them, one of the lab coats crunched when I smoothed it out.
I tracked down the culprit: one of the pockets was harbouring an (extremely) old latex glove that was falling apart. As I was explaining what the source of the crunch was to the technician, I exclaimed that it looked rather terrible and it was “old and crunchy.”
At which point, it could be subsequently heard in my head being voiced by my good colleague Tom still back in Buffalo, “Like your mom, yeaaaaaahhhhhh!” Despite that immaturity, still one of the most rational & logically minded individuals I know. And a great sounding board when I need it.
1Having worked with tritium in the past, I sort of loathed radioactivity; no convenient means by which to detect it. Thankfully, 32P is way easier to detect & monitor! So I can go home and not be worried about sucking radioactive wing sauce off of my fingers to mutagenize my gonads & germ layers, 2-week half-life or not.
2These lab coats are very likely older than the undergraduate students in the lab, and likely threatening to be older than myself, I’d hazard.
Every scientist gets lulled into a false sense of security, even by the most unlikely of scenarios. The extent varies with respect to each individual’s level of safety/accident paranoia, but I feel confident in saying it happens to all of us. I was reminded this evening of how the innocuous can easily become the dangerous.
So, in a slight change of events, instead of the typical personal blog posts I’ve been doing lately, I feel a need to make a more technical one. Either for others’ benefit or for my own, in the event I happen to lose said notes on how to do this stuff. In any case, these are the summation of online searches, trial & error, and a bit of ingenuity in other random spots, all directed towards the generation, editing, and mapping of surface electrostatics maps on structural models of crystallized proteins. I’ll drop in cited links when I can, pending whether or not I can track some of them down again!