I have been working on purifying a 16kDa His-tagged protein for the past year (literal year -- began this project on May 6, 2016). I finally refined my protocol to the point that I was getting brilliantly pure product. The protein itself is exceptionally finicky, but alas, progress!
I'll begin from my elution step -- After my washes, I elute my protein using a buffer composed of 50mM sodium phosphate, 650mM NaCl, and 7% glycerol (pH 8.0). I dialyze my protein in a 50mM sodium phosphate, 100mM NaCl, and 3% glycerol buffer (pH 8.0).
I have attempted lyophilizing my protein twice. In my first attempt, I filled 2ml tubes with 1.5ml of my sample, snap froze it, perforated the caps of the tubes, put them into a vacuum flask and set it up with the LabConco FreeZone 2.5 lyophilizer. I went back the next day to find a goopy mess.
For my second attempt, I had consulted the professor in charge of the machine and he recommended 1. reducing my sample volume per tube and using a larger tube overall, 2. freezing on a slant, and 3. covering the tube top with two layers of kim-wipes secured by an elastic as opposed to capping and perforating. Given this advice, after dialysis, I split my samples into two 50ml falcon tubes with 6ml of sample in each tube. I snap froze them in liquid nitrogen on a slant and stored them on dry ice. I turned on the lyophilizer to allow it to cool and have the vacuum reach optimal pressure. In this time, I set the vacuum flask that I was using in dry ice to cool it so that everything would be as cold as possible. Once the machine was ready, I loaded my two tubes into the frozen vacuum flask and applied it to the lyophilizer. After opening the vacuum port, I watched to make sure that there was no thaw and for the 30 minutes that I watched, frost had vanished and there was no visible thaw/melt. I left it in there overnight and came back to see that it had become a fairly small volume of viscous goop with white specks of powder interlaced in the goop. I am immediately reminded of the frosting that comes with Pillsbury toaster strudels.
I am unsure as to how to move forward from here per preparing my samples. I am open to recommendations and protocols that anyone might have that can help. This is a most disappointing step to be failing at given the laborious task of purifying the protein, so I hope I can get a quality, powdery, lyophilized product soon!
Thank you!
It's the glycerol. It is not volatile, so it is remaining in the sample. It is a liquid, so it is impossible to prepare a solid sample by lyophilization of a glycerol-containing solution. If you must add a polyol stabilizer, use sucrose or trehalose, which are solids. Better yet, do not lyophilize the protein at all. Just store the aliquots at -80oC.
Hi Adam, I just remade some elution and dialysis buffer without glycerol. Hoping it works!
If possible exchange the buffer to a minimum condition with the least amount of salt.
Because of the intended downstream applications, a lyophilized product is preferred so that we can measure and make particular concentrations of the protein for in vivo and in vitro work.
I am in the process of purifying my protein and will begin dialysis tomorrow afternoon. Currently, glycerol is present in my sonication buffer, but I have removed it from my binding, wash, elution, and dialysis buffers. My dialysis buffer is 25mM PBS, 85mM NaCl, and 15mM sucrose. I will do three buffer changes during dialysis over the course of 2hr/2hr/overnight.
I plan on slant freezing ~6ml in a 50ml falcon tube for lyophilization. Is this ideal? Does anyone have any recommendations on how I might improve preparation for lyophilization at that specific step? A postdoc recommended including dry ice in my vacuum flask with my sample so as to keep everything as cold as possible, but I am hesitant to do this because I don't know how it will behave in a vacuum.
It should not be necessary to include dry ice. I would recommend insulating the 50-ml tube from contact with the glass flask using some styrofoam. Make sure your lyophilizer is keeping a hard vacuum.
I've seen someone line the vacuum flask with foil as well. In any case, I will bring along the styrofoam rack that falcon tubes are shipped in. I'll keep everyone posted on my progress. Really hope it works this time!
I am also wondering -- what is the ideal vacuum pressure? The LabConco manual and the protocol I saw online from a lab at Harvard say ideally, add samples once it's at ~0.133 mBar, but I've read online that it should be well below 0.1 mBar -- otherwise it'll just be evaporating the sample slowly as opposed to freeze drying.
The moment you open the valve connecting the lyophilizer to your flask, the vacuum pressure will go way up because of the air in the flask, so it doesn't matter too much what the vacuum pressure was just then. What matters is that the pump is working well enough to bring the pressure back down quickly and keep it there.
Hi Adam, following all of your recommendations, my protein lyophilized beautifully. I am testing for bioactivity on Monday, so hopefully it isn't all just the PBS/NaCl from my dialysis buffer!
Be careful when you redissolve the lyophized material with water to add back a similar volume to what was removed, not much less. Otherwise, the salts will be very concentrated, which could either denature or precipitate the protein.