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