I have previously been using a high pressure cell disruptor, but it's currently unusable until we get a replacement part. In the mean time, I'm trying to purify an enzyme out of E. coli and last minute am borrowing protocols/materials from the lab down the hall. They have a bead beater that I can use but they work with yeast so only have 0.5 mm glass beads. I know it's not ideal and that I should use 0.1 mm glass beads for bacteria, but beggars can't be choosers. Any suggestions how to make the best out of what I've got right now? Should I increase/decrease homogenization time and/or ratio of beads to sample? I'd love to hear some ideas before I just wing it :) Thanks!
People have already used 0.5 mm glass beads for E coli cell disruption.
Please check out this article
(Biotechnology and Bioprocess Engineering
October 2008, Volume 13, Issue 5, pp 613-623)
You can easily use 0.5mm beads with bead to sample ratio of 1:5 and 5 cycles of 30sec ON and 20sec OFF unless it is a membrane bound protein.
One can use glass beads for cell disruption (0.5 mm; vortex 8x for 20s, with intermediate keeping the eppis on ice for 1 min); but you should be aware that it might change the properties of your expressed protein. We have tested in our lab different cell disruption methods and luckily in our case the protein did not show huge differences in stability or binding to resin properties.
If you have problems to purify the protein after cell disruption with glass beads, maybe you have the possibility to grind your cells with precooled mortar&pestle under liquid nitrogen. This is a very mild cell disruption.
One can use glass beads for cell disruption (0.5 mm; vortex 8x for 20s, with intermediate keeping the eppis on ice for 1 min); but you should be aware that it might change the properties of your expressed protein. We have tested in our lab different cell disruption methods and luckily in our case the protein did not show huge differences in stability or binding to resin properties.
If you have problems to purify the protein after cell disruption with glass beads, maybe you have the possibility to grind your cells with precooled mortar&pestle under liquid nitrogen. This is a very mild cell disruption
One can use glass beads for cell disruption (0.5 mm; vortex 8x for 20s, with intermediate keeping the eppis on ice for 1 min); but you should be aware that it might change the properties of your expressed protein. We have tested in our lab different cell disruption methods and luckily in our case the protein did not show huge differences in stability or binding to resin properties. If you have problems to purify the protein after cell disruption with glass beads, maybe you have the possibility to grind your cells with precooled mortar&pestle under liquid nitrogen. This is a very mild cell disruption
You should be able use the glass beads but the efficiency with glass beads might not be as good as with a sonicator or pressure cell disruptor. I think you should be able to use lysozyme (0.1-0.2mg/ml final concentration) to disrupt cells by stirring the lysate at moderate speed in a coldroom (4 deg C) for 10-12 hours. And then use the glass beeds to reduce the viscosity of the lysate due to mostly DNA. If you have access to Sonicator, I would suggest to go for sonicator in the first place.
Good Luck.
Venu.
Thanks everyone! I actually had to purify before anyone had responded, but it turned out good enough for what I needed this time around. Yield was way better than using a broken cell disruptor and activity was exactly what we expected. I decided to go crazy with it and try a ratio of 1.5g beads to 1ml cell suspension. I had about 10ml total divided into 6 tubes so that each was only 1/2 full. I used a bead beater for 20 min total 3 tubes at a time switching resting on ice in 2 minute intervals. I will take your advice for next time to not beat for so long. It sounds like my way was overkill but luckily the enzyme survived. Thanks again!
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