Protein Crystallization

...

Solution A: 15% w/v PEG 4000, 1M NaCl in 50 mM NaAc, pH 4.5

Solution B: 20% w/v PEG 4000, 1M NaCl in 50 mM NaAc, pH 4.5

Solution C: 25% w/v PEG 4000, 1M NaCl in 50 mM NaAc, pH 4.5

Solution D: 30% w/v PEG 4000, 1M NaCl in 50 mM NaAc, pH 4.5

Using the hanging drop method, 5 µL of protein solution was mixed with 5 µL of the reservoir solution and placed in the crystal wells as shown below:

1+A

1+B

1+C

1+D

2+A

2+B

2+C

2+D

Nail polish was applied around the top of the wells to seal the crystallization wells upon placement of the cover slips. The hanging drop solutions were placed on the cover slips before putting, drop-side down, on top of the corresponding crystallization/well solutions. Nail polish was again applied on the edges of the slips to secure them. For the next 7 days, the set-ups were observed under a stereo microscope to monitor crystal growth.

- Results and Discussion

- Conclusion

The optimum conditions for protein crystallization using NaCl and PEG as precipitants were determined through the hanging drop method. Results have shown that ___mg/mL lysozyme in _____ resulted in the most satisfactory crystal growth.

It is recommended that the set values for the parameters, e.g. temperature and amount of reagents be strictly followed as deviations from the stated values can significantly alter the recrystallization procedure.

- References

Chayen, N. & Saridakis, E. (2008). Protein crystallization: from purified protein to diffraction-quality crystal. Nature Methods, 5(2), 147-153. http://dx.doi.org/10.1038/nmeth.f.203

Durbin, S. & Feher, G. (1996). PROTEIN CRYSTALLIZATION. Annual Review Of Physical Chemistry, 47(1), 171-204. http://dx.doi.org/10.1146/annurev.physchem.47.1.171

Lorber, B., Skouri, M., Munch, J., & Giegé, R. (1993). The influence of impurities on protein crystallization; the case of lysozyme. Journal of Crystal Growth, 128(1-4), 1203-1211. http://dx.doi.org/10.1016/s0022-0248(07)80124-2

McPherson, A. (2004). Introduction to protein crystallization. Methods, 34(3), 254-265. http://dx.doi.org/10.1016/j.ymeth.2004.03.019