I have tried to lyse organoids for protein extraction using RIPA with protease and phospatase inhibitors. I found that from 5 pooled organoids my yield was low (1-2ug/ul). Has anyone tried another lysis buffer or have they have any success with Ripa? Or are the organoids not good for protein extraction in the first place!?
Any help would be much appreciated
Hi Jessica, I don't know if this helps, but I worked with mouse F9 teratocarcinoma cells a long time ago.
I treated them with retinoic acid and PMA to make them form endoderm structures.
The cells balled up and I had to spin them out and extract the proteins in SDS buffer using syringes with small bore needles to repeatedly suck in and expel the lysate to shear the DNA.
It worked out fine for protein determination and blots.
Hi Jessica,
I am working with PDAC Organoids and most often I get reasonable amount of protein concentration. Based on my experience, the type of buffer doesn't have an impact on the final protein concentration, but what matters is the amount of wells which you harvest and the confluency of the organoids. I normally harvest 6 wells of 24-well-plate and the organoids are at least 70% confluent. Furthermore try to get rid of the matrigel by sonicating the organoids mixed with the buffer for 30 seconds.
Good luck and best wishes,
Zahra
Hi Zahra Dantes,
Can you please share your protocols with me or you can just recommend the papers regarding it?
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