Dear all,
I made cell lysate of Chondrocytes (Primary Cells) using the Lysis Buffer given below -
10mM Potassium Phosphate Buffer
1mM DTT
500mM Sucrose
100mM Ny.aF
Protease Inhibitor
After adding the lysis buffer, I sonicate the cells and then spin at 13000 rpm for 10 min and then collect the supernatant and use for the catalase assay.
But, when I did protein estimation I am getting very low amounts of protein (0.0616 ug/ul). I tried lysis thrice, but all the times I had the same result. I am confused as to where the things are going wrong.
Should I use a different lysis buffer or a different procedure (than just sonication)?
The protein concentration depends on the volume of buffer and the amount of cells. It may be that your lysis went OK but the cell suspension was very dilute. You could try it with a lower volume.
Alternatively, your sonication may have been ineffective (or both).
One way to find out is to test your supernatant for catalase activity. If you see none, but expect it to be there, then you will have reason to believe that the sonication was ineffective. Try to improve the technique, or find another way of breaking open the cells.
There's a very high concentration of sucrose in your lysis buffer. The high viscosity and hyperosmolarity may be reducing the effectiveness of the sonication. Try reducing the sucrose concentration. Add a freeze-thaw step to help break the cells.
Can you describe your sonication procedure in detail?
What is Ny.aF?
Sorry it was a typo... It is Sodium Fluoride (NaF).
> After scraping the cells, we centrifuge them at 2000 rpm for 3min to get the pellet.
> Then, we discard the supernatant and add 1ml PBS and spin at 13000 rpm for 5min.
> Discard the supernatant and add lysis buffer by keeping the microfuge (containing cell pellet) tube on ice.
> Then, in ice we sonicate for 6 cycles of 3sec sonication and 20sec gap
> After sonication- we spin at 13000 rpm for 10 min at 4oC.
> After the spin, we save the supernatant and use for enzyme assay. At this step we aliquot the supernatant in to smaller volumes and store at -4oC for short term storage and at -85oC for long term storage. But, when we aliquot the lysate we directly take from the microfuge tube and aliquot.
I have doubt whether I should be collecting the supernatant separately and resuspend it and then aliquot?
I'm not clear on what you are asking. It makes sense to me to do what you are doing: storing aliquots of the supernatant. But you should make sure the enzyme is stable to this treatment. Test this by comparing the enzyme activity in the fresh supernatant to the activity in the 4oC-stored and frozen supernatants.
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