Preparing Cell Lysates

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Related Resources: Brochures | Application Notes

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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
• 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

Possible Cause	Remedy
Insufficient buffer/tissue ratio	Use less tissue and/or more buffer. If using dissociated, cultured cells, make sure cell counts are accurate. Get tips on accurate cell counting using the Scepter™ handheld counter Scepter™ cell counter ordering information

Variability in Measured Total Protein Concentration Between Replicates

Possible Cause	Remedy
Inaccurate pipetting	Ensure accurate pipetting; use properly calibrated pipettes.
Incorrect protein standard selected (for colorimetric assays)	Switch to infrared-based protein quantitation, which directly measures amide bonds in protein chains Learn about infrared-based protein quantitation
Time before reading absorbance too long (for colorimetric assays)	Process fewer samples at once. Switch to infrared-based protein quantitation, which is not time-dependent. Learn about assay-free infrared-based total protein quantitation using the Direct Detect® system
Assay incompatible with buffers used	Verify that buffers used are compatible with chosen protein quantitation assay. Use infrared-based protein quantitation, which is compatible with a wider range of buffers than colorimetric assays. Learn about infrared-based protein quantitation of cell lysates
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.

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

Possible Cause	Remedy
Pipetting error resulting in incorrect dilution of standards.	Ensure accurate pipetting; use properly-calibrated pipettes. Explore Direct Detect® infrared-based protein quantitation system, featuring single-time standard curve generation per sample buffer.

Degradation Band on Blot

Possible Cause	Remedy
Protein degradation due to insufficient protease inhibition	Add or increase concentration of protease and phosphatase inhibitors. Try using different inhibitor cocktails. Explore inhibitor cocktail options

Bands Smeared Vertically

Possible Cause	Remedy
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	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

Bands of Interest Obscured