I am working on Yeast. Lysing the Cell in Phosphate buffer in Cell disruptor. My Protein of intrest is not stable at high temperature because of which activity loss observed. Can anyone suggest me any stabilizer which can help me to stabilize my POI at high temperature during lysis..
Which lysis protocol you use?
It is not better you changed your lysis protocol?! until find an stabilizer?!
My preferred method of lysis when preservation of molecular structure is desired is to resuspend yeast in your lysis buffer (usually 0.5mL per gram of cells) and then drip them into liquid nitrogen. They should form individual droplets.
To lyse them, grind the cells in a mortar and pestle that has been chilled with liquid nitrogen until they become a fine powder. Move the powder into a new clean tube. It can be stored at -80 for months. When you want to use the lysate, thaw slowly at 4 degrees. After 5-10 minutes, you can add some fresh lysis buffer (usually 1mL per gram) and some acid-washed beads. As the tube thaws, vortex every 5-10 minutes until it is liquid.
To clarify lysate, spin once at 3000-5000g for 5min. Transfer supernatant to a new tube and then spin again for 10 min between 10,000 and 20,000 g.
I use this protocol to get functional ribosomes from cells.
What temperature are you use? The most important things are temperature and contact time.You can use vacuum to decrease tempareture at lysis.
M. Yu and Max ferretti, Lysis was done at 4-10oC (Cold condition) in cell disruptor. I think heat generated due to friction (inside the diamond of cell disroptor) might be resulting in activity loss. I can not change the whole lysis buffer or the instrument. So need to find a stabilizer which can help to absorb heat shock, generated inside the lysis chamber or any chemical which can make the cell wall more soft so i can decrease the lysis pressure hence the friction and temperature.
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