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Preparing Cell Lysates

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Related Resources: Brochures | Application Notes
Before you start analyzing a protein sample by Western blotting, read our web page, “Protein Preparation,” for technical and product information on this important part of the Western blotting workflow. 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

Merck:/Freestyle/BI-Bioscience/Protein-Detection/western-blotting/WB-troubleshooting/protein_conc_too_low.jpg
Protein concentration too low as seen on Coomassie-stained membrane
Possible CauseRemedy
Heat degradation
  • Perform all lysate preparation steps at 4°C.
  • If sonicating, minimize total sonication time or insert breaks in between sonication pulses to minimize heat generation.
Protein degradation
  • Add or increase concentration of protease and phosphatase inhibitors.
  • Try using different inhibitor cocktails.
  • Explore inhibitor cocktail options
  • Create lysate aliquots to minimize number of freeze-thaw cycles.
Inadequate tissue homogenization
  • Increase time/power setting of sonication.
Cell debris contamination
  • When pipetting supernatant following centrifugation, take care not to disturb the pellet.
Too little tissue/too few cells
Wrong type of lysis buffer

Merck:/Freestyle/BI-Bioscience/Protein-Detection/western-blotting/WB-troubleshooting/sample_pH_too_low.jpg
Effect of excessively low sample pH on SDS-PAGE
 Different proteins may be best visualized via Western blot using different lysis buffers. Consider the pH, buffering compound, detergents and other additives when determining the optimal buffer for a specific protein. Order lysis and extraction reagents:
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Protein Concentration Too High

Possible CauseRemedy
Insufficient buffer/tissue ratio
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Variability in Measured Total Protein Concentration Between Replicates

Possible CauseRemedy
Inaccurate pipetting
  • Ensure accurate pipetting; use properly calibrated pipettes.
Incorrect protein standard selected (for colorimetric assays)
Time before reading absorbance too long (for colorimetric assays)
Assay incompatible with buffers used
Protein concentration outside of linear dynamic range of assay
  • Increase or decrease lysate concentration until it is within the linear dynamic range of the selected assay.
  • Choose an assay with a broader linear dynamic range.
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Non-Linear Standard Curve (for Colorimetric Assays for Total Protein)

Possible CauseRemedy
Pipetting error resulting in incorrect dilution of standards.
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Degradation Band on Blot

Possible CauseRemedy
Protein degradation due to insufficient protease inhibition
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Bands Smeared Vertically

Possible CauseRemedy
Protein degradation
  • Increase concentration of protease and/or phosphatase inhibitors during cell lysate preparation.
  • Avoid repeated freeze/thaw cycles of cell lysate and Western blot samples.
Incompatibility between lysis buffer component and SDS

Effect of interfering detergents (right lane) in SDS-PAGE
Effect of interfering detergents (right lane) in SDS-PAGE
 
  • Cationic detergents in the lysis buffer will modify charge of SDS micelles, causing smearing. Remove or substitute these detergents.
  • Nonionic detergents at high concentrations will modify SDS micelles, causing smearing. Dilute, or remove these detergents.
Excess concentration of abundant proteins, such as albumin, in the sample
  • First deplete the protein sample of abundant proteins, using magnetic or agarose beads designed for depletion and/or enrichment
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Bands of Interest Obscured

Bands of interest on SDS-PAGE obscured by abundant proteins
Bands of interest on SDS-PAGE obscured by abundant proteins
Possible CauseRemedy
Excess concentration of abundant proteins, such as albumin, in the sample
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