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I tried to extract polysomes fraction from human osteoblast...
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Annalisa Frattini Apr 5, 2018
I tried to extract polysomes fraction from human osteoblast (primary culture) but I obtained a very low quantity of polysomes and proteins.
I belive that the lysis buffer is not appropriate for these cells and I am not able to optimally lysate the cells.
Do you have any suggesti Read more
I belive that the lysis buffer is not appropriate for these cells and I am not able to optimally lysate the cells.
Do you have any suggesti Read more
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Amaresh Chandra Panda Author Answered Apr 8, 2018
Institute of Life Sciences Bhubaneswar
Thanks for the query. There are 2 modifications which helped us get a good amount of polysomes from primary cells using the same polysome extraction buffer (PEB).
Suggestions:
1. Replating the cells 1-2 days prior to polysome fractionation increases the amount of polysome and higher polysome peaks in primary cells using the same PEB.
If this does not help, please try the following along with suggestion 1.
2. Lyse the cells with Dounce homogenizer using the PEB supplemented with a higher amount of RNase inhibitor.
Hope this helps,
Amaresh
Suggestions:
1. Replating the cells 1-2 days prior to polysome fractionation increases the amount of polysome and higher polysome peaks in primary cells using the same PEB.
If this does not help, please try the following along with suggestion 1.
2. Lyse the cells with Dounce homogenizer using the PEB supplemented with a higher amount of RNase inhibitor.
Hope this helps,
Amaresh
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