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	48-602MAG	Buffer Detection Kit for Magnetic Beads	1 Kit

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Preparing Cell Lysates

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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

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 Cause	Remedy
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	Use more tissue and/or less 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
Wrong type of lysis buffer 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. Learn about cell lysis and protein extraction solutions Order lysis and extraction reagents: Bacterial lysis and extraction Mammalian cell lysis and extraction

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 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

Bands of Interest Obscured

Bands of interest on SDS-PAGE obscured by abundant proteins

Possible Cause

Remedy

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
Learn more about fast, easy depletion of abundant proteins using PureProteome™ magnetic beads
Order PureProteome™ magnetic beads

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