Protein Extraction Efficiency of OMNI Bead Ruptor Elite Bead Mill Homogenizer and Detection of Total and Phosphorylated Proteins by HTRF® Immunoassay

Protein extraction techniques from organs, tissues, and cells is a critical step for validation of protein expression in biomedical research, such as in drug efficacy and toxicity studies. Upstream of protein extraction, sample preparation is required to lyse cells within a sample, releasing analytes of interest into solution for downstream evaluation. Traditional lysis methods can be inefficient and time consuming, typically involving manual hand-held homogenizers or enzymatic techniques. The Omni Bead Ruptor Elite™ bead mill homogenizer, however, has been shown to be a quick and efficient method for sample preparation of various types like tissues and cells [1]. 

In this application note, MCF7 cells, an estrogen receptor positive breast cancer cell line, were cultured and treated with MAPK/ERK pathway inhibitors (U0126 and PD98059) or treated with a cell cycle (CDK4/6) inhibitor, Palbociclib. The cells or cell pellets harvested from the tissue culture plates were lysed using two methods: the Omni Bead Ruptor Elite bead mill homogenizer and standard cell lysing method described in the protein detection kits. The resulting cell lysate samples were tested for total cellular protein content and expression of total and phosphorylated ERK and Rb (retinoblastoma) proteins to determine the protein extraction efficiency of the two methods of lysate sample preparation, as well as the effects of inhibitor treatments. Results indicated that the Omni Bead Ruptor Elite bead mill homogenizer is a quick and effective sample preparation instrument when extracting proteins from cells.

This was demonstrated through the detection of total cellular protein, total and phosphorylated ERK and Rb proteins and the inhibition of phosphorylated ERK and Rb protein expressions in cells treated with inhibitors.

Figure 2: Total protein extraction efficiency of Omni Bead Ruptor Elite bead mill homogenizer, comparing to lysing the cells for 60 min on a plate shaker. Total protein levels were measured using BCA protein assay kit.