The lab has been trying to express a particular type of fusion protein in bacteria, and this series somehow tended not to make it to the end of the first Ni-NTA elution in one piece. They seem to like to fall apart into two-three pieces. We did everything right otherwise (protease inhibitors, 4C, fast protocols, etc). I am not a protein prep guru, but this time the degradation seemed excessive.
So I figured... what the hell, let's try to do the entire initial prep, including NiNTA elution, in a -20 room. We have one at the Institute, and it's never used for anything exciting anyway.
Not sure if it worked yet, but the experience was interesting. Not sure if what I did was right, but here are my observations so far.
I started out by preparing 1 gallon of 50% glycerol in a styrofoam container. I placed a metal eppendorf holder in it and pre-chilled it in the -20 room for a day. This was supposed to be a -20 bath, in case I decided to bail from the -20 room into a 4C room nearby. In hindsight, this turned out to be a very good idea, except for the 50% glycerol bath. Not sure why I decided to keep it liquid, a block of ice precooled to -20 would have done the job just fine. I also could have combined this with dry ice on the bottom, but I did not want my solutions to freeze.
Placed a spare beaker of 50% glycerol, about 2L, next to the main bath. That turned out to be useful for cooling the sonicator tip -- again, in hindisght.
Placed a centrifuge, a shaker, and a sonicator into the -20 room. Checked them next morning, everything seems to switch on. Verified the temperature of the -20 bath, it was indeed -20. Kept the sonicator controller outside and ran the cable inside (did not want condensation to ruin the main board of the controller).
Prepared a lysis buffer based on 50% glycerol, pre-chiled it to -20, it remained liquid. Prechilled about 20 eppendorves in the -20 bath. There was no lysozyme, as this is -20, so I figured it was useless anyway.
Washed NiNTA agarose beads with the buffer in batch mode. Spinning worked fine, agarose beads settled fine at 1000 g. Found that, when at -20, the NiNTA beads are not keen to be resuspended by flicking, only pipetting up and down with the 1 ml tip worked.
I got myself a down parka with a hood and a hat and a pair of ski gloves. Good luck opening eppendorved in thick gloves. This is the first time I wished I had one of those eppendorf-opening tools.
I had about 500 ul of 4 cell pellets in 4x50 ml falcons. I thawed the pellets to 4C (as they were still in PBS, that would never resuspend at -20), then mixed with 1 ml of the -20 lysis buffer, placed it all in the -20 bath. In this case, the fact that the glycerol remained liquid, helped. If it had been a block of ice I would not be able to dip the falcons with cells in it.
The next step was done in the -20 room, where I brough a 1ml pipet and a box of 1 ml tips as well. It started looking like a small polar research lab.
Atttempted to resuspend the cells in 1 ml of lysis buffer by pipetting up and down. That did not go well (expected) -- everything was viscous and snotty as hell. Blasting it with 6W of ultrasound helped: after this I managed to transfer most of the stuff into fresh eppendorves, it was till snotty from the genomic DNA. In hindsight, 1 ml of 50% glycerol-based lysis buffer was too little, too viscous, and unmanageable. 2 ml would have made it better, and I could probably still fit everything into 2 ml large eppendorves.
I blasted each eppendorf with the lysates 4 times more with30" and 6W of ultrasound, to the point they got non-snotty. by that time, my fingers were numb, and I was not enjoying myself. Holding eppendorves with gloves was mostly OK, but opening them not so much, so I kept one hand bare, and that almost resulted in frostbite.
Interestingly, the sonicator did not mind being continously on at 6W at -20, I guess the temperature sensor does not go off. So I just kept it on (the controller was oustide) and held eppendoves one by one, with the tip inside. Gradually, the solutions cleared, so I could see the tip inside. Cooled the tip by dipping in 50% glycerol bucket between eppendroves -- that came quite handy.
After 15 min of this I was getting concerned about my fingers freezing, so I placed the 4 tubes in the centrifuge. Adjusting the time to 15 min was fun: my fingers kept freezing to the buttons. I managed to start it, and then got the hell out of there to warm my hands. But the most annoying step was done. Not sure if it stayed at -20 inside from the heat of spinning, should have tested with a tube with water (if ice remains ice, it was -20).
In hindight, 6 blasts would have been better, or using 10W. I am saying it, b.c. after spinning I still got ~50% of unlyzed cells.
It took two spins to clear the solution. I transferred the -20 bath into the 4C room. It went up to -15 in about 20 min, not bad. Mixed about 500 ul of lysates with 100 ul of washed Ni beads and left on the shaker (rocker, to be exact) overnight. Transferred the -20 bath back into the -20 room.
Will see what I've got next day. It was not too bad, after all. Having one solid -20 ice bath, one liquid 50% glycerol -20 bath, and one beaker of clean 50% glycerol for sonicator wash would have been ideal. Also, next time, if this is ever done again, I should use 2 ml of the lysis buffer for this: the lysates get extremely gooey and non-manageable. Not sure if this can be done in larger volumes, unless one splits 20 ml of lysate into 10 eppendorves (larger centrifuges do not get to -20).
Here is a picture of the -20 bath in the 4C room (the latter felt nice and warm compared to the -20). The lowest reading on the thermometer is -10, and it's another 8C below that. The block holes for tubes are shallow, but they are eppendorf-shaped (I have a custom drill bit for that). Since my NiNTA beads were only 100 ul in volume, they stayed nice and chilly.
The level of glycerol went down with cooling... not sure if it's the shrinkage, or if there is a leak in the goddamn styrofoam plastic bucket. Actually, therre are two containers: the plastic styrofoam is inside of a bigger styrofoam one in which I filled the gaps with packaging peanuts and sealed with Alu tape on top.
Update 2 days later: washing, elution etc worked great. Washed 100 ul of beads with 1 ml of 50% glycerol-based buffer, 4 times, eluted with 150 ul of 500 mM Imidazole, got about 1 mg/ml of all proteins, in 150 ul, from about 50 ul of culture. Clean, no signs of degradation at all.
Will try to repeat with more challenging proteins.