Begin by chemically modifying the pooled protein fractions through reductive methylation. To do so, take the purified protein and add 20 millimolar dimethylamine borane, followed by 40 millimolar formaldehyde to it. Incubate the mixture in oscillatory shaker for two hours at four degrees celsius.
Then, add an additional 10 millimolar dimethylamine borane to the mixture. Next, incubate the mixture overnight for 12 to 18 hours at four degrees Celsius. Quench the reaction by adding 100 millimolar tris chloride having a pH of 7.5.
Use the appropriate wash buffer and elution buffer for loading the methylated protein onto a nickel-NTA column. First, wash the two milliliter nickel-NTA column with 10 column volumes of wash buffer. And then, elute the protein with the elution buffer.
Following elution, pass the protein eluate through a PD-10 desalting column, pre-equilibrated with the appropriate buffer. Add cholesterol prepared in isopropanol or ethanol to the desalted protein, and incubate the mixture overnight at four degrees Celsius. The next morning, ultracentrifuge the mixture at 150, 000G for 10 minutes at four degrees Celsius.
Add the collected supernatant to a centrifugal concentrator with a 100 kilodalton cutoff, and concentrate the supernatant to a final concentration of 30 to 50 milligrams per milliliter. Once more, centrifuge the concentrated protein using a high speed benchtop refrigerated centrifuge, and remove the pellet. Place the collected supernatant on the ice at four degrees Celsius before proceeding to set the crystallization.
Alkylated proteins stored at four degrees Celsius were analyzed by analytical gel filtration chromatography over the course of a month. And it showed a slight protein loss after a week.
