Western Blot Troubleshooting | Antibody Company, Buy Antibodies Online
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 Protocol?
Here are the 5 most common problems our customers run into their WB experiments
Western Blot Troubleshooting Weak Signal
Weak signal is typically caused by problems in the blocking or washing steps, but can also be caused by a number of other issues. Use these tips to identify and troubleshoot you weak signal results.
Keywords: Weak Signal, Low Signal, Western Blot Troubleshooting
Western Blot Troubleshooting Bands Wrong Molecular Weight
Western blotting separates proteins based on size; large proteins migrate through a polyacrylamide gel matrix slower than small proteins do. However, other factors can influence the migration rate of proteins, resulting in band sizes different than predicted based on protein size alone.
Keywords: Band Size, Wrong Band Size, Western Blot Troubleshooting
Western Blot Troubleshooting Background Background Blotchy, Flecked, Or Dirty
Many Western blot problems arise due to issues with the background. Splotches, streaks, or background staining can ruin results. Use these troubleshooting tips to identify and resolve the cause of your background troubles.
Keywords: Flecked Background, Speckled Background, Blotched Background, Western Blot Troubleshooting
High background on a western blot occurs when the background signal of the membrane reduces the signal-to-noise ratio to unreadable levels. Use these troubleshooting tips to identify and resolve the cause of your high background.
Keywords: High Background, Saturated Background, Background Staining, Nonspecific Staining, Western Blot Troubleshooting
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 these tips.
Keywords: Smile Bands, Distorted Bands, Uneven Bands, Western Blot Troubleshooting
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. You will most likely you will find your problem in here.
Issues Related To 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
Issues Related To 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
