I have purified a recombinant M0 AB(1-42) using a recent method from Article A Robust and Efficient Production and Purification Procedure...
I have used 15mM NaOH as SEC buffer and also tried to keep it in monomeric state by bath sonicating for 1 minute. I have also tried filtration of these peptides using 10 kDa cut off centricon. I put my peptides for aggregation after adding it in pH 7.0 sodium phosphate buffer and maintaining the pH of the initial peptide solution to 7.0 and initial concentration to 50 micromolar. I find out concentration of abeta using absorbance at 293 nm and extinction coefficient 2400 mol L-1 cm-1 . I set the amyloid peptide solution at 37 degree Celsius and 200 rpm in orbital shaker incubator. Still the solution is not showing any turbidity and also no increase in thioflavin-T fluorescence even after 72 hours. I also obtained circular dichroism spectra and I am getting only random coil in my peptide solution as implied by the spectra even after 48 hours. I just observe thread like entities floating in the solution but no increase in turbidity. Please help me out with this problem. I suspect amorphous aggregate formation.
Your thread-like entities sound reminiscent of microbial contamination. You could try adding sodium azide (~ 0.02%) to your buffer, and/or sterile filtering all buffers and solutions before using. Otherwise, you would be able to distinguish amorphous vs amyloid aggregation by electron microscopy. Good luck.
Dear Vanessa,
The protein solution was syringe filtered. All buffers were autoclaved and filtered before use. The MCTs were also sterile. So there shouldn't be a problem of microbial contamination. Still we could try sodium azide to be sure
Dear Vanessa,
I have tried everything possible Still i can't get any aggregation. I checked by EM and it seems to form amorphous aggregates. What could be the reason for non aggregation into amyloid fibrils for such a high propensity amyloidogenic peptide. I am in desperate need of help. Please help me out.
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