Western Blot Troubleshooting

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

    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

    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