I followed the available protocols, link given, to purify cytoplasmic ribosomes from HeLa cells. After the ultracentrifugation, ribosome pellet was washed briefly 3-4 times. However, I can see the presence of actin in western blot, alongwith ribosomal proteins.
Is it fine to get actin along with the ribosome pellet or I am contaminating my ribosome prep with other cytoplasmic proteins??
http://www.ncbi.nlm.nih.gov/pubmed/21154551
I only got ribosomes as a side-product when extracting membranes but what I remember is that they showed up as a very clear, very (!!!) sticky pellet in the tube, which were hard to loosen and resuspend. Therefore I wonder if your washes are thoroughly enough, if you write about 3-4 brief washes. Did you resuspend the pellet completely for washing? I don't think you will loose much material by adequate washing.
Hello Vivica,
Thank you for the reply. After getting the tight and sticky pellet as you also mentioned, I did not resuspend and wash them, rather washed as pellet attached to the tube with PBS.
Can we say that it is the cytoplasmic actin which is coming somehow (and not the actin from on going translation, I did not use any translation inhibitor as wanted active ribosomes), and washing is improper? of course I shall try washing after resuspending them,
How about using nucleases to destroy the mRNA of the ungoing translation? Or you use translation inhibitors once in a preparation and look if there's a big difference in actin content? Then you might know where the actin comes from. As I was never interested in the ribosomes, I did not care much about purity of the preparation.
For translation assays one might use commercial reticulocyte lysate or wheat germ extract (both available at Promega).
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