I'm trying to obtain a clear supernatant after homogenising a 5% dried cell weight (packed cell wet weight = 15%) yeast suspension. However, after centrifuging this in a Beckmann centrifuge, the supernatant is quite cloudy and contains a lot of 'gunk' which makes it difficult to filter. Can someone advise how I can get around this problem?
Hi Michael,
Do you need the extract for? to purify a protein/s, or other?
What is the buffer that you have used, and what is the extraction method?
How did you clarify it, centrifugation at what g/speed and for how long? Is the extract viscous or not?
In many cases turbid extract contains nucleic acids that were not precipitated during the clarification (they also usually add viscosity to the extract), and if proteins is what you need they may interfere with following steps.
To this end, you may need to add another step to remove those from the extract. Different methods at different steps can be employed such as: DNaseI treatment, PEI treatment, ammonium sulfate precipitation, LiCl precipitation, chromatography, and more...).
Hope that this an initial lead for you. Will be glad to help more once you send more details.
Best,
Ghil
Ghil Jona Yes I need the extract to obtain a protein-rich and particle-free solution to carry out some precipitation studies. We have used a 10mM phosphate pH7.5 buffer to re-suspend dried yeast, followed by cell breakage using a high-pressure homogeniser operating at 500 bar pressure for a total of 5 passes with cooling. The homogenate was then centrifuged at 18,000xg for 45 minutes to obtain the supernatant. The supernatant was by no means clear and showed some viscosity.
The ammonium sulphate precipitation step you mentioned I have seen somewhere so this could be a starting point.
Many Thanks
Michael
Glad that it is helpful.
A remark for you: given that Ammonium sulfate precipitates also proteins, I would consider to use DNase I. Another thing that could help is to include some salt in your buffer: 100-200mM NaCl, or KCl should also improve the extract, and help precipitate some of the nucleic acids.
A couple of additional questions:
1. was the pellet firm or quite loose?
2. could you observe at the perimeter/edge of the pellet some orange translucent precipitate? If yes that translucent pellet is composed of nucleic acids.
Best of luck
The pellet was quite firm and seemed to consist of multiple layers with distinct colours. I don't quite remember seeing an orange translucent precipitate though. Also we could see a waxy, gelatinous-like precipitate on the surface of the liquid.
Thank you for your help!
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