Hello Luminaa Anandh

You will have to use a non-denaturing lysis buffer since you will be assessing the activity of an enzyme. The non-denaturing buffer will contain mild non-ionic detergent and a combination of various salts and agents to enhance extraction and stability of proteins. Non-ionic detergent like NP-40 or Triton X-100 are surfactants, basically soaps, that disrupt the fatty acid bilayer. NP-40 can be used to solubilize proteins but not denature them. The non-denaturing lysis buffer should also contain low salt concentration, usually 120 mM NaCl. Some salt is needed to prevent non-specific protein interactions. EDTA which is a common additive that has multiple functions including protease inhibition and protection against oxidative damage may be added to a final concentration of 5mM. Tris is another additive used to buffer the pH and prevent protein denaturation. It is always better to add protease inhibitors in the lysis buffer prior to use in order to protect proteins.

You may use the following recipe for preparing non-denaturing lysis buffer.

NP-40/Triton X-100 lysis buffer:

50 mM Tris HCl, pH 8.5,

120 mM NaCl,

5mM EDTA,

1% detergent (NP40 / Triton X-100).

However, the concentration of salt and detergent could be altered according to your needs. Also, please note that protease inhibitors should be added fresh to the lysis buffer at the time of lysis.

Hope this helps!

Good Luck!

Hi Luminaa Anandh

There are several lysis buffer options that can be used for lysing Urine Kidney Epithelial Cells to assess enzyme activity. Some commonly used lysis buffers include:

The choice of lysis buffer will depend on the type of protein of interest, its location within the cell, and the downstream application. It is recommended to optimize the lysis buffer conditions for the specific cell type and protein of interest to achieve optimal protein yield and activity.