I tried to extract polysomes fraction from human osteoblast...

protocol Protocol: Polysome Fractionation to Analyze mRNA Distribution Profiles
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 Read less more less
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
1 answer
Sort by: most helpful / most recent

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
0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer