Problems related to Microsome Preparation

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benzenefancy
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Problems related to Microsome Preparation

Post by benzenefancy » Tue Jun 30, 2009 7:21 pm

I would like to find the answers related to my questions in separation by centrifugation as I am troulbed by the absence of results!!!

I used LCC6MDR to take microsomes from fragement of plasma membrane. I lysed the cells by lysis buffer first and then centrifuged it at 500g for 10 min in cold room to take supernatant use another centrifugation at 5000g for 10 min in cold room. Then I loaded 2mL of the lysate to 3mL 35%sucrose cushion with dropper. I found that some drops distrubed the sucrose layer surface during the loading process. However, I found that there was still two liquid layers so that I continued the experiment.

After 1hr centrifugation at 100000g in cold room, there were no two layers!!! The protocol I used claimed that microsome is in the tubrid interface between upper supernatant layer and the sucrose cushion layer!!! Can anyone tell me what may be possible reasons for my results and provide some suggestions for me to finish this work? And I would like to know how to prevent disruption of the sucrose layer when I add solution onto it. Also, what is the best way (skill) to take the tubrid interface containing microsome? Thx!!!

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