Hello everyone,
I am trying to isolate antimicrobial peptides from plants. After precipitating the peptides with acetone, most of the pellet (peptides) did not dissolve. I used dH2O/PBS to dissolve the pellet. I do not want to use SDS or urea to dissolve the pellet because it will affect my cell culture experiment.
This is my peptides isolation protocol:
-The plant material was ground in a blender. For extraction, 10% CH3COOH (Acetic Acid) in the presence of a commercial proteinase inhibitor was added to the floured plant parts (added to the ground material at a ratio of 1:10 (w:v)).
- The extraction was carried out with continuous vigorous stirring for 1 h at room temperature. After 1 h, mixture was sieved; fine particles were separated by centrifugation at 4700 rpm for 10 min. The supernatant was filtered through a Whatman paper filter.
-Cold acetone (-70 °C) was poured into the filtrate at a ratio of 1:7 with gentle stirring; the mixture was then kept at +4 °C for overnight.
-After this time, the suspension was centrifuged at 4700 rpm for 10 min. The supernatant was removed and the resulting fraction was dried at room temperature. The dried precipitate was redissolved in dH2O or PBS.
Try dissolving it in dimethylsulfoxide (DMSO). This is a water-miscible solvent that is generally very useful for this sort of thing, and it is compatible with subsequent biological experiments when diluted to a few % by volume.
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