Hi Kumar, 1) While you contemplate other ideas, have you tried the following already? - microseeding or macroseeding with the tiny crystals you have? - crystallization using sitting drop vapour diffusion under oil? Often, one gets a burst of nucleation with several tiny crystals. Not sure if this is what you see. Oil can retard the process favourably if the crystals are in a hurry.
2) If you are not already familiar, I recommend reading the foll. paper to learn about how to design DNA ends for crystallization. ---- Tan S, Hunziker Y, Pellegrini L, Richmond TJ. Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization. J Mol Biol. 2000 Apr 7;297(4):947-59 ---- It is often necessary to design and screen a whole bunch of DNA lengths as well as DNA ends for protein-DNA crystallization. 3) Also, while you consider gel filtration, another alternative is preparative gel electrophoresis with polyacrylamide gel matrix. Will require some set up and optimization but worked very well for me before. Having said that, I have seen some folks getting well diffracting crystals with no purification following mixing protein and DNA. Not everyone gets that lucky though. Wish you good crystals. Raji ---------Included Message---------- >Hi, >I am trying to crystallize a protein-DNA complex. I purify the protein finally >using gel filtration. I purchase >single stranded complementary oligos (desalting from idtdna.com), mix them up >and make DNA duplex by >heating to 95 degree C and cooling to room temperature. I mix protein and DNA, >concentrate and use it >for crystallization. >I am geting small crystals consistently under a specific condition. These >crystals take up IZIT dye but are >not well shaped. I am not able to improve the size and shape of the crystals >substantially even after >screening with additives (Hampton research). >I suspect that purity of the duplex DNA (presence of unpaired oligos) is >limiting the chances of obtaining >better crystals. > >How can I purify the duplex DNA further? > >Are there better ways of making protein-DNA complex for crystallization? > >If I make the protein –DNA complex and then do the gelfiltration, will >the >complex purified so be a better >choice for crystallization? > >Thank you >Kumar > >Dept. of Biochemistry, Cellular and Molecular Biology, >Walters Life Science, # 406, >University of Tennessee, TN, Knoxville, USA > > ---------End of Included Message----------
