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Western Blotting Troubleshooting

Pro tips on resolving common Western Blot issues such as weak signal, wrong band size, smiley gel, and high background.

How to Troubleshoot Western Blot

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

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High Background

High background on a western blot occurs when the background signal of the membrane reduces the signal-to-noise ratio to unreadable levels. Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.

S.No. Possible Cause Solution
1 Antibody concentration is too high
  • Optimize and decrease antibody concentration
  • Use a dot-blot test to optimize 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, increase blocking solution concentration, 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 Exposure time is too long
  • Check exposure parameters and 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
  • Never re-use blocking solutions
  • Use pure protein as a blocking agent
17 Contaminated equipment
  • Ensure all equipment and tools are clean and no gel is left on membrane
  • Use disposable incubation trays
  • Thoroughly wash reusable incubation trays between incubations
18 Insufficient antibody binding activity
  • Use fresh aliqot of antibody stored at -20°C
  • Avoid thawing and re-freezing antibodies
  • Store antibodies at -80°C for long-term stability
19 Excessive substrate incubation
  • Reduce length of substrate incubation
20 Blocking proteins reacting with detection system
  • Milk contains biotin; do not use when detecting with avidin-biotin system
  • Try one of Boster's other blocking reagents

Weak/No Signal

A weak western blot signal is characterized by faint or indistinct bands. While the bands may be barely visible at their predicted sizes, weak signal can often require repeating the experiment. Common sources of this error occur during blocking, washing, protein transfer, or detection. Use the tips below to identify the source of the error and get better results.

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
  • Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent.
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
  • Use a dot blot assay to optimize protein concentration
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
25 Insufficient sample concentration
  • Increase the amount of starting material
  • Concentrate your sample using immunoprecipitation or similar procedure
26 Transfer too vigorous
  • Reduce transfer time or voltage to prevent small proteins transferring completely through membrane
  • Use a secondary membrane to capture proteins transferred through the primary membrane
  • Use a membrane with smaller pore size
26 Inadequate transfer
  • Increase transfer time or voltage
27 Sandwich assembly oriented incorrectly
  • Make sure the sandwich assembly is oriented correctly relative to the electric field
  • Check the polarity of the electric field
28 Incorrect transfer buffer pH
  • Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio
29 Insufficient antibody binding affinity
  • Reduce washing stringency
  • Increase antibody concentration
  • Use Boster high affinity primary antibodies
30 Insufficient sample loading
  • Use more starting material
  • Concentrate sample prior to loading

Blotchy, Flecked, Or Dirty Background

The background of a western blot does not always appear clean and flawless. Blotches, streaks, and spots are all common artifacts that can make it hard to interpret and publish your results. These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer. Follow the tips below to identify and solve the cause of your imperfect western blot background.

S.No. Possible Cause Solution
1 Blotched background: Uneven antibody distribution
  • Agitate during incubation to coat the membrane evenly in incubation buffer
2 Blotched background: Membrane dried out unevenly
  • Make sure membrane is thoroughly wetted before starting the protocol
  • Ensure the membrane does not dry out during any step
3 Blotched background: Uneven wash/incubation buffer coverage
  • Increase volume of wash and incubation buffers
  • Do not stack membranes during incubation
4 Flecked background: Secondary antibody aggregation
  • Increase secondary antibody dilution to prevent aggregation
  • Spin down or filter out antibody aggregates
5 Flecked background: Clumps of blocking buffer binding secondary antibody
  • Use fresh blocking buffer
  • Increase Tween 20 concentration of blocking buffer
  • Filter blocking buffer before use
  • Use a different blocking reagent, such as albumin, BSA, or casein
  • Wash membrane with wash buffer before antibody incubation
6 Flecked background: Buffer contamination
  • Mix new buffers
  • Filter buffers before use
7 White spots with no protein transfer: Air bubbles trapped between gel and membrane during transfer
  • Carefully squeeze out bubbles from between membrane and gel using a sterile glass rod
  • Use enough buffer to saturate the membrane

Wrong Band Sizes

Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.

S.No. Possible Cause Solution
1 Bands have higher MW than expected: Proteins are glycosylated or bear other post-translational modifications
  • Review literature and identify modified forms of your target protein
  • Strip post-translational modifications with enzymatic treatment
2 Bands have much higher MW than expected: Protein aggregation
  • Decrease protein concentration
  • Prepare new sample with fresh loading buffer
3 Bands have much higher MW than expected: Incomplete denaturation or residual disulfide bonding
  • Denature the protein with urea
  • Use stronger reducing agents
  • Use fresh 2-mercaptoethanol or DDT to strip disulfide bonds
4 Bands have lower MW than expected: Protein sample has been digested or degraded
  • Use fresh sample from frozen stock
  • Use a lysis buffer with proteinase inhibitors
5 Bands have lower MW than expected: Primary antibody is detecting splice variants
  • Review literature to identify splice variants of your protein
  • Try a different primary antibody
6 Bands have lower MW than expected: Primary antibody binding a similar epitope on a different protein
  • Run a negative control to detect other proteins that react with your antibody
7 Multiple bands: Primary or secondary antibody contaminated with nonspecific IgG
8 Multiple bands: Nonspecific binding of primary antibody
  • Increase antibody dilution
  • Affinity purify primary antibody to select for only desired binding activity
  • Use Boster primary antibodies guaranteed to only bind their indicated targets
9 Multiple bands: Nonspecific binding of secondary antibody
  • Reduce antibody concentration
  • Run a negative control with just the secondary antibody to detect nonspecific binding
10 Multiple bands: Insufficient blocking
  • Use higher concentration blocking buffer
  • Block for longer
  • Add Tween 20 to blocking buffer
11 Multiple bands: Ionic interactions
  • Increase stringency of washing step
  • Increase salt concentration of incubation buffers
  • Include stronger detergents in the washes

Distorted Bands

Distorted bands can make it very hard to interpret your results. Common distortions include smile-shaped bands with the edges trailing upward, diffuse bands that are broad or blurry, and streaked bands that trail off in several directions. Make sure your next blot has even, crisp bands by following the tips below.

S.No. Possible Cause Solution
1 Curved, "smiling" bands: Electrophoresis voltage too high
  • Reduce voltage; run the gel slower for more consistent results
2 Curved, "smiling" bands: Overheated gel
  • Reduce voltage or run the gel on ice or in a cold room (-4°C)
3 Streaked or diffuse bands: Incomplete contact between gel and membrane during transfer
  • Use thicker filter paper in sandwich
  • Squeeze air bubbles and excess buffer from between membrane and gel
4 Streaked or diffuse bands: Slippage of membrane during transfer
  • Avoid agitating or moving the gel or membrane during transfer
5 Blurry bands: Electrophoresis voltage too high
  • Run the gel for longer at a lower voltage
6 Blurry bands: Improper loading buffer composition
7 Blurry bands: Air bubbles trapped between gel and membrane during transfer
  • Carefully remove bubbles by squeezing them out with a sterile glass rod
8 Ghost bands: Overexposure during visualization
  • Decrease the exposure time
9 Ghost bands: Loading sample too concentrated
  • Reduce the amount of sample loaded
10 Ghost bands: Antibody concentration too high
11 Ghost bands: Blot was moved during transfer
  • Avoid agitating or moving the gel or membrane during transfer
12 Uneven, crooked bands: Poor gel polymerization
  • Check gel concentration and ensure complete dissolution of SDS before casting gels
13 Uneven, crooked bands: Varying salt concentration between wells
  • Make sure the salt concentration in different samples is similar

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