Western blot remains one of the most popular immunoassay techniques for its capacity to provide researchers with a quick, visual representation of their sample. Yet, it can also be one of the more difficult methods to troubleshoot, and a protocol adaptation that improves chemiluminescent western blot data may not be similarly applicable to fluorescent detection. This article lists some common western blot problems and suggests ways of addressing them to achieve more reproducible results.
According to Kenneth Oh, Ph.D., Global Senior Product Manager, Applications and Collaborations Protein Quantitation and Imaging Business Unit at Bio-Rad Laboratories, western blot problems fall into several categories. These include low target protein band intensity, high background, and the presence of multiple bands and/or indiscreet band patterns. Smeared lanes and saturated bands are other frequent complaints. “Such issues can lead to the protein band of interest being incorrectly identified or quantified, translating to inaccurate interpretation of results,” he says. “Additionally, a poor western blot image can mean the expense of having to repeat an experiment or can cause researchers to give up on a set of conditions or reagents that seemingly failed but could have been successful with optimization.”
“A western blot image is far more than just a pretty picture unless a simple yes/no answer is needed,” reports Kristi Ambroz, Senior Director of Science and Global Support, Biotechnology, at LI-COR Biosciences. “Researchers undertaking these types of experiments typically want to quantify their data, making it essential to apply strategies for increasing reproducibility. A tried and trusted approach is to perform direct detection using fluorescence instead of chemiluminescence, which eliminates the need for a highly variable enzymatic reaction. Also, optimizing the amount of protein loaded—whether that be the target of interest or a protein used for normalization—is crucial to ensure the amount of protein detected is proportional to the signal on the blot.” Other methods for improving reproducibility focus on using an appropriate imaging system and data analysis software and are included in the table below.
While troubleshooting strategies will vary depending on whether a western blot employs chemiluminescent or fluorescent detection, there is considerable overlap between the two methods. The following table comprises some common western blot complaints and potential fixes, with any application-specific issues being noted in parentheses.
Confirm that an appropriate sample type was used by referring to sites such as UniProt, PAXdb, or proteinatlas.org, and to antibody manufacturers’ datasheets, for information about protein expression
Determine the appropriate amount of sample to load by conducting a linear range experiment
Enrich the target protein through immunoprecipitation, protein precipitation, or sub-cellular fractionation
Prepare fresh samples
Include protease inhibitors in the lysis buffer, and phosphatase inhibitors if performing phosphoprotein detection
Keep samples on ice prior to adding loading buffer
Avoid freeze-thaw cycles
Match the lysis buffer to the target; whole cell lysates can often be prepared using a buffer containing NP-40, while extracting cytoplasmic or nuclear proteins may require using Triton X-100 or RIPA buffer, respectively
Check the transfer was performed in the right direction
Pre-wet nitrocellulose membranes in PBS or TBS, and PVDF / LF-PVDF membranes in 100% methanol, followed by PBS or TBS, prior to transfer
Confirm successful protein transfer by Ponceau staining the membrane or acquiring a Stain-Free image if using Stain-Free gels and a Stain-Free enabled imager
Use a total protein stain such as REVERT™ Total Protein Stain to check for even transfer across the membrane
For low molecular weight proteins, increase the methanol concentration in the transfer buffer to 30–40%, reduce the transfer time and voltage, and use a membrane with a smaller pore size (0.2 μm instead of 0.45 μm)
For larger proteins (>140 kDa), reduce the methanol concentration in the transfer buffer to 10%, consider adding 0.05% SDS to the transfer buffer, and increase the transfer time
After transfer, rinse the blot in buffer (with no Tween® 20) and dry completely before moving to blocking
Avoid blocking for longer than 1 hour
Perform blocking at room temperature (not in a 37oC incubator)
Check whether the antibody recognizes native or denatured protein
Run a positive control sample, such as a lysate known to endogenously express the target protein, an over-expression lysate, or a recombinant protein to verify antibody performance
Check that primary antibodies are validated for the chosen species
Confirm that primary antibodies are validated for western blot
Switch to using alternative primary antibodies
Try using a different blocking buffer or optimizing blocking conditions
Titrate primary and secondary antibodies to determine optimal concentrations (consider using tools such as the Mini-PROTEAN II Multiscreen Apparatus)
Use fresh antibodies for each experiment (do not re-use antibodies)
Increase the duration of the primary antibody incubation step
Increase the antibody volume to ensure the entire membrane surface is covered with liquid and will not dry out
Gently agitate the membrane during antibody incubation steps
Check the expiry dates of antibody reagents
Confirm antibodies have been stored correctly
Avoid reusing antibodies
Confirm that the correct secondary antibody was used (e.g., if the primary antibody was raised in rabbit, an anti-rabbit secondary is required for detection)
Increase the exposure time
Consider using an imaging system that provides automated exposure or measures photon rate such as the Odyssey® XF, Odyssey® M, or the ChemiDoc™ family of imagers
Always store fluorophore-conjugated antibodies away from light
Check the expiry date of chemiluminescent detection substrates
Use fresh detection reagents/substrates for every experiment
Switch to using a more sensitive detection substrate, such as Clarity Max™
Ensure buffers do not contain azide
Confirm that fluorophores are compatible with the imaging system and that the imaging system is set to read the correct wavelengths
Enrich the target protein through immunoprecipitation, protein precipitation or sub-cellular fractionation
Refer to sites such as UniProt for information about protein isoforms
Check whether isoform-specific primary antibodies are available
Refer to sites such as UniProt for information about PTMs such as phosphorylation, glycosylation, and cleavage
Switch to using different primary antibodies
Perform a blocking peptide experiment to distinguish between specific and non-specific bands
Test the two secondary antibodies separately before combining them to understand the expected staining pattern for each
Use secondary antibodies from the same host species to avoid potential cross-reactivity
Avoid using mouse and rat primary antibodies together, or sheep and goat primary antibodies
Use cross-adsorbed secondary antibodies, such as IRDye secondary antibodies
Reduce the concentration of secondary antibodies
Check fluorophore properties to avoid spectral overlap
Confirm that fluorophores are compatible with each other and the chosen imaging system
Use an imager with LED-based detection, such as the ChemiDoc MP™, or laser-based detection such as the Odyssey® family of instruments
Consider using a rapid blocking buffer such as EveryBlot Blocking Buffer
Consider using a non-mammalian protein-based blocking buffer such as Intercept® Blocking Buffer or Intercept® Protein Free Blocking Buffer
Include the blocking agent in antibody diluents
Avoid detergents (Tween® 20) in the blocking agent
Reduce the duration of the primary antibody incubation step
Include up to 0.2% Tween® 20 in the primary antibody diluent
Avoid using milk for blocking, especially when working with anti-goat secondary antibodies
Include up to 0.2% Tween® 20 in the secondary antibody diluent
If using a PVDF membrane and fluorescent detection, include 0.01% SDS in the secondary antibody diluent
Run secondary antibody-only controls
Increase the number, volume, and/or duration of wash steps
Include up to 0.2% Tween® 20 in antibody diluents and wash buffers
Ensure the blot is covered in buffer at all times
Switch to using a nitrocellulose membrane
Consider using a low-fluorescence PVDF membrane such as Trans-Blot Turbo RTA LF-PVDF or Immobilon®
Decrease the exposure time
Consider using an imaging system that provides automated exposure or measures photon rate such the Odyssey® XF, Odyssey® M, or the ChemiDoc™ family of imagers
Try using a detection substrate with a different sensitivity
Always wear gloves when handling the membrane
Avoid scratching the membranes with forceps
Ensure blotting apparatus is thoroughly washed and dried after use—clean transfer pads and transfer boxes by soaking in 100% methanol for 10 minutes and use 100% methanol to clean any trays used for incubations
Prepare fresh buffers for every assay or store buffers short-term at 4oC if appropriate
Use a roller to gently remove any air bubbles from between the gel and the membrane when setting up the transfer
Filter the blocking buffer before use
Switch to using a different blocking buffer
Incubate each membrane in a separate tray
If incubating membranes together is unavoidable, ensure sufficient volumes are used for all membranes to move freely
Clean the scanning surface with an alcohol-based cleaner before imaging
After transfer, rinse blot in buffer
Allow the dye front to migrate completely through the gel
Switch to using a loading buffer that does not contain blue dye
Load less sample
Reduce the duration of antibody incubation steps
Reduce the voltage
Run the gel in a cold (4oC) room
Check the expiration date of precast gels
Switch to using a different lysis buffer
Try sonicating samples or heating for longer
Centrifuge samples prepared in running buffer before loading onto the gel
Include DNase in the lysis buffer
Use thicker filter paper and/or more sponges in the sandwich
Be consistent with incubation temperatures and timings
Confirm you are operating within the linear dynamic range
Switch to using direct fluorescent detection
Optimize the amount of sample loaded to ensure all targets and normalization proteins are detected in a manner proportional to the signal on the western blot
Check that sample treatments do not impact the expression of proteins used for normalization
Ensure a consistent method is used for sample preparation
Perform Total Protein Normalization as an alternative to housekeeping protein normalization
Increase the number of replicates for more statistically relevant results
Always perform data analysis using an unaltered digital image
Consider using data analysis software designed to minimize operator errors (e.g., Empiria Studio™)