Preparing Cell Lysates

Often, the first step of analyzing protein expression or protein-protein interactions is to obtain a cell or tissue sample, lyse the cells, and extract proteins using extraction reagents. Total protein concentration must be determined for these cell lysates. Variables affecting each of these steps are outlined below, as each could affect the sensitivity and reproducibility of the Western blot.

Click on the symptoms to read about the possible causes and remedies:

• Total Protein Concentration Too Low
  
• Protein Concentration Too High
  
• Variability in Measured Total Protein Concentration between Replicates
  
• Non-Linear Standard Curve (for Colorimetric Assays for Total Protein)
  
• Degradation Band on Blot
  
• Bands Smeared Vertically
  
• Bands of Interest Obscured

Total Protein Concentration Too Low

Protein Concentration Too High

Variability in Measured Total Protein Concentration between Replicates

Non-Linear Standard Curve (for Colorimetric Assays for Total Protein)

Degradation Band on Blot

Bands Smeared Vertically

Bands of Interest Obscured