Western Blot Troubleshooting

Troubleshooting guides

Troubleshooting guides

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The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the Western blot assays.

  1. High Background


    S.No. Possible Cause Solution
    1 Too high antibody concentration
    • Optimize and decrease antibody concentration
    2 Aggregate secondary antibody formation
    • Filter the secondary antibody through 0.2μm filter
    • Use a new secondary antibody
    3 Too high antibody incubation temperature
    • Incubate the antibody at 4°C
    4 Non-specific secondary antibody binding or cross-reactivity with blocking agent
    • Run secondary antibody control (without the primary)
    • Decrease secondary antibody concentration
    5 Cross-reactivity of primary or secondary antibody with blocking agent
    • Add Tween-20 to the incubation and washing buffer
    6 Incompatible blocking agent
    • Compare different blocking buffers
    7 Incomplete blocking
    • Optimize choice of blocking buffer
    • Increase protein concentration in blocking agent
    • Optimize blocking time and/or temperature; Block for 2 hours at normal temperature or overnight at 4°C
    • Add 0.05% Tween 20 detergent into blocking agent
    • Add 0.05% Tween 20 detergent into antibody diluents solution
    8 Insufficient blocking
    • Extend blocking time or use a compatible blocking agent (e.g. skim milk, BSA, serum, etc.)
    9 Cross-reactivity of antibody with other proteins
    • Use different blocking agent (Do not use skim milk with biotin system
    • Reduce secondary antibody concentration
    • Test cross-reactivity between secondary antibody and membrane
    10 Insufficient washing
    • Increase number of washes and buffer volume
    • Add 0.05% Tween 20 detergent into washing buffer
    11 Too long exposure time
    • Reduce exposure time
    12 Membrane problem
    • Use clean tweezers; Operate with gloves
    • Use new membranes
    • Ensure the liquid is enough to keep the membrane moist
    • Use decolorization table in incubation
    • Avoid membranes overlapping
    • Handle carefully and avoid damaging membrane
    13 Insufficient membrane wash
    • Increase the number of wash
    14 Incompatible membrane
    • Nitrocellulose membrane’s background is lower than that of PVDF membrane
    15 Dry membrane
    • Make sure membrane is covered with a sufficient amount of liquid and prevent it from drying
    16 Contaminated buffer
    • Use new buffer or filter buffer before use
    17 Contaminated equipment
    • Ensure all equipment and tools are clean and no gel is left on membrane
  2. Weak/No Signal


    S.No. Possible Cause Solution
    1 Improper protein transfer to membrane
    • Stain gel after transfer is complete to determine transfer is efficient
    • Use Ponceau S to stain membrane to determine transfer is efficient
    • Ensure sufficient contact between gel and membrane during transfer
    • Make sure transfer sandwich is assembled correctly
    • Wet membrane according the instruction
    • Avoid overheating during electro-transfer
    • Use positive control or molecular weight markers
    • Optimize transfer time and current
    • Use Boster’s Membrane-Transferring Buffer (AR1149)
    • Avoid sample (antigenic determinant) destroy when handling
    2 Insufficient protein and membrane binding
    • Adding 20% methanol to transfer buffer
    • Use small-bore membrane
    3 Insufficient antibody
    • Increase antibody concentration
    4 Insufficient antigen
    • Load more protein
    5 Antigen masking by blocking buffer
    • Compare different blocking buffers
    • Optimize protein concentration of blocking agent
    • Reduce blocking time
    6 Presence of sodium azide in buffers
    • Eliminate sodium azide from buffers
    7 Too short exposure time
    • Lengthen film exposure time
    8 Too short substrate incubation time
    • Lengthen substrate incubation time to five minutes
    9 Digestion of protein on membrane
    • Optimize amount of blocking agent
    10 Degradation of protein during storage
    • Re-prepare protein sample
    11 Incompatible primary and secondary antibodies
    • Make sure primary antibody, secondary antibody, substrate, enzyme system and samples are compatible
    • Use loading control to test effectiveness of second detecting system
    12 Low concentration of primary antibody and/or secondary antibody
    • Increase antibody concentration
    • Increase incubation time
    13 Cross-reactivity between blocking agent and antibodies (primary or secondary)
    • Use mild detergent such as Tween20
    • Change blocking agent (commonly used are milk, BSA, serum or gelatin)
    14 Inability of primary antibody to recognize the protein in tested sample
    • Check instruction
    • Use positive control
    15 Low or none content of target protein (ineffective antigen)
    • Use positive control
    • Increase loading amount to 20-30 µg protein per well
    • Use protease inhibitor or fractional extract target protein
    16 Insufficient transfer and excessive wash
    • Check the transfer with Ponceau S
    • Soak PVDF-membrane in methanol
    • Avoid excessive wash
    17 Over-blocking
    • Use 0.05% skim milk or no milk diluents buffer
    • Change blocking agent
    • Reduce blocking time
    18 Loss of primary antibody effectiveness
    • Prepare fresh antibody and store properly when not in use
    • Avoid repeated freezing and thawing
    19 Inhibition of secondary antibody by sodium azide
    • Avoid using sodium azide together with HRP- conjugated antibodies
    20 Loss of effectiveness in enzyme conjugate and substrate
    • Mix enzyme conjugate and substrate (no color development when enzyme is inactive)
    • Use activated enzyme conjugate and fresh substrate
    21 Improper wet transfer for membrane
    • Soak PVDF membrane in 100% methanol
    22 Insufficient molecular weight of target protein (< 10 kDa)
    • Use small-bore membrane
    • Reduce transfer time
    23 Equality or nearness in values between target protein’s isoelectric point and transfer buffer’s pH value
    • Try other buffers such as CAPS buffer (pH 10.5)
    • Try low pH value buffers such as acetic acid buffer
    24 Too high methanol concentration
    • Decrease methanol concentration or use isopropyl alcohol