Western Blot Analysis: Complete Protocol, Results Interpretation & Quantification Guide
Western blot analysis (immunoblotting) is a cornerstone technique in molecular biology that lets you detect and semi-quantify specific proteins in a complex sample. This guide covers the full western blotting workflow from sample preparation through image analysis, with practical guidance on reading results, using ImageJ for densitometry, and avoiding the most common experimental mistakes.
What Does a Western Blot Tell You?
Western blot purpose and what you can detect
A western blot answers one fundamental question: is a specific protein present in my sample, and roughly how much of it is there? Because proteins are separated by molecular weight before detection, you also get size information — helping you confirm the correct isoform, detect post-translational modifications (PTMs), and identify unexpected cleavage products or complexes.
Protein Presence
Confirm whether a target protein is expressed in your cell line, tissue, or lysate.
Relative Expression
Compare protein levels across conditions, time points, or sample groups by band intensity.
Protein Size
Estimate molecular weight in kDa using co-run molecular weight markers.
PTM Detection
Detect phosphorylation, glycosylation, or cleavage via size shifts or PTM-specific antibodies.
Protein Interactions
Combined with immunoprecipitation (co-IP), verify binding partners in a complex.
Disease Biomarkers
Detect aberrant protein expression or cleavage associated with disease states.
Western Blotting Principle
How the technique works from first principles
The western blotting principle rests on three sequential physical events: size-based separation (SDS-PAGE), immobilisation (transfer to membrane), and specific detection (antibody binding + signal generation). Each event depends on the preceding one, so a failure in any stage propagates downstream.
SDS (sodium dodecyl sulfate) denatures proteins and imparts uniform negative charge proportional to chain length. During electrophoresis, smaller proteins migrate faster through the polyacrylamide mesh. After transfer to a nitrocellulose or PVDF membrane, proteins are immobilised in the same spatial pattern as in the gel, but now accessible to antibodies. A specific primary antibody binds the target; a labeled secondary antibody amplifies and reports the signal. The position of the resulting band, compared with molecular weight markers, gives size; the intensity of the band is proportional to protein abundance within the linear detection range.
Step-by-Step Western Blotting Protocol
Western blotting steps explained with practical guidance
Sample Preparation
Lyse cells or homogenise tissue in an appropriate lysis buffer containing protease (and phosphatase, if needed) inhibitors. Quantify total protein using a Bradford or BCA assay. Dilute all samples to the same concentration and denature in SDS-PAGE loading buffer at 95°C for 5 minutes.
Critical: equal loadingSDS-PAGE Electrophoresis
Load denatured samples and molecular weight markers into polyacrylamide gel wells. Apply voltage (typically 80–120 V). Negatively charged SDS-protein complexes migrate toward the anode; separation is primarily by molecular weight.
Principle: size-based migrationProtein Transfer to Membrane
Transfer resolved proteins from the gel onto a nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane using electrophoretic blotting (wet tank or semi-dry). Verify transfer completeness by Ponceau S staining of the membrane or by staining the residual gel.
Key choice: NC vs. PVDF| Property | Nitrocellulose (NC) | PVDF |
|---|---|---|
| Protein binding | ~80 μg/cm² | ~170 μg/cm² |
| Background | Low | Slightly higher |
| Stripping & reprobing | Moderate | Excellent |
| Low MW proteins | Good | Better (<20 kDa) |
| Pre-wet with methanol | Not required | Required |
| Best for | Standard WB, ECL | Fluorescent, reprobing |
Blocking Non-Specific Binding
Incubate the membrane with a blocking agent (5% non-fat dry milk or 3–5% BSA in TBST) for 1 hour at room temperature. Blocking saturates free protein-binding sites on the membrane, preventing antibodies from binding non-specifically and causing high background.
Goal: low-noise western blot imagesPrimary and Secondary Antibody Incubation
Incubate the blocked membrane with a diluted primary antibody specific to your target protein (typically overnight at 4°C). After washing, incubate with a species-matched secondary antibody conjugated to HRP or a fluorophore.
Specificity determines result reliabilityDetection Method Selection
Choose a detection chemistry matched to your sensitivity requirements and imaging equipment.
Method choice affects quantification accuracy| Method | Principle | Sensitivity | Quantitative? | Equipment |
|---|---|---|---|---|
| ECL (Chemiluminescent) | HRP oxidises luminol; emits light | ~1–10 pg | Semi (linear range) | Film or CCD imager |
| Fluorescent | Fluorophore-conjugated secondary | ~1–10 ng | Yes (wide dynamic range) | Fluorescent imager |
| Chromogenic | HRP precipitates coloured substrate | ~10–100 ng | Limited | White light scanner |
| Radioactive | ⁺P or ⁻S isotope-labeled | High | Yes | Phosphorimager |
Image Acquisition
Capture the membrane image using X-ray film, a CCD-based digital imager (e.g., Bio-Rad ChemiDoc), or a fluorescent scanner. For quantitative analysis, always use a digital imager and save in a lossless format (TIFF or uncompressed TIF). Avoid JPEG, which introduces compression artefacts that corrupt densitometry measurements.
Save as TIFF for quantificationWestern Blot Results Interpretation
How to read western blot results accurately
Interpreting western blot results requires evaluating three dimensions simultaneously: band position (molecular weight), band intensity (abundance), and band pattern (number and sharpness of bands). A confident interpretation requires a positive control, a negative control, and molecular weight markers in every experiment.
Interpreting Unexpected Band Sizes
Not every band at an unexpected molecular weight is an artefact. Common explanations include:
- Post-translational modifications (glycosylation, phosphorylation) add mass above the theoretical size.
- Incomplete denaturation or disulfide bonds that survive boiling leave oligomers or complexes.
- Splice variants or isoforms of the same gene run at different sizes.
- Proteolytic cleavage of a precursor produces a product smaller than predicted.
- Cross-reactivity of the antibody with a structurally related protein.
Western Blot Quantification with ImageJ
Densitometric analysis, normalisation, and data presentation
Densitometric analysis converts the visible band on your blot into a numerical intensity value that can be compared across lanes. ImageJ (freely available from NIH) and commercial alternatives such as Image Lab (Bio-Rad) or Fiji are the most widely used tools. The process follows four sequential steps to yield valid, normalised data.
- Verify the linear range
Before quantifying, confirm your bands were captured within the detector's linear range. Run a serial dilution of your lysate, blot and plot density vs. loading amount. Only work within the linear portion. Saturated bands cannot be quantified accurately.
- Open image and set up lanes in ImageJ
Open your TIFF image in ImageJ. Go to Analyze → Gels → Select First Lane, then draw a rectangular selection around the first lane. Advance to each subsequent lane using Select Next Lane. Maintain a consistent selection size across all lanes.
- Plot and measure band area (densitometry)
Select Plot Lanes to generate an intensity profile for each lane. Use the straight-line tool to close the baseline of each peak, then the wand tool to click inside each peak and record its area. Export values to a spreadsheet.
- Subtract background
Use a region of the membrane without any protein to measure local background. Subtract the background value from all band measurements.
- Normalise to loading control
Divide each background-subtracted band intensity by the loading control band intensity in the same lane (e.g., actin, GAPDH). This corrects for lane-to-lane loading variation.
- Calculate relative expression and present data
Express normalised values relative to a reference condition (set as 1.0). Calculate mean and standard deviation across biological replicates. Plot as bar charts with error bars and apply appropriate statistical tests.
Common Pitfalls and How to Avoid Them
The most frequent sources of failed or unreliable western blot results
- High background / non-specific bandsCaused by insufficient blocking, over-incubation with antibody, or dirty wash steps. Fix: increase blocking time, optimise antibody concentration, and increase wash stringency.
- No signal or very faint bandPrimary or secondary antibody too dilute, incomplete transfer, or target protein not expressed. Check transfer efficiency with Ponceau S before antibody incubation.
- Saturated bands — over-interpretation riskSignal intensity is non-linear once saturation is reached. Always verify the linear range before making quantitative comparisons.
- Unequal loading without loading controlEven a 20% difference in loaded protein produces a visually convincing intensity difference. Always normalise to a loading control before interpreting band intensity differences.
- Antibody cross-reactivityAn antibody may bind proteins with similar epitopes. Always validate with a KO or knockdown control.
- Using milk to block for phospho-antibodiesMilk contains casein (a phosphoprotein) that competes with phospho-epitopes. Use BSA or commercial phospho-blocking buffers instead.
- Saving images as JPEG for quantificationJPEG compression introduces artefactual pixel intensity changes. Save all images for quantification as uncompressed TIFF files.
- Interpreting band size without marker alignmentPre-stained markers can shift position slightly. Always run markers in the same gel and align them carefully when estimating molecular weight.
Decision Guide: When to Use Western Blot Analysis
Choose the right technique for your experimental question
Western blot analysis is the method of choice in several scenarios, but it is not always the optimal tool. Use the scenarios below to decide whether western blotting or an alternative technique better fits your experimental question.
You need size confirmation
You want to verify that the detected protein is the correct molecular weight, distinguishing it from isoforms or degradation products.
Comparing relative expression
You need to show that a protein is up- or down-regulated between two or more conditions and can tolerate semi-quantitative data.
Detecting PTMs
Using phospho-specific or modification-specific antibodies to ask whether a protein is modified under your experimental conditions.
High-throughput protein quantification
You need to quantify dozens of proteins across hundreds of samples. ELISA, mass spectrometry, or protein arrays are more scalable.
Subcellular localisation
If you need to know where in the cell the protein resides, immunofluorescence (IF) or immunohistochemistry (IHC) are more informative.
Protein–protein interactions
Immunoprecipitate a complex, then use western blot to confirm co-precipitated binding partners (co-IP western blot).
Advantages and Limitations of Western Blotting
Making informed decisions about when and how to apply the technique
- Detects specific protein in a complex mixture
- Provides molecular weight information
- Requires relatively small sample amounts
- Can detect PTMs with modification-specific antibodies
- Membrane can be stripped and re-probed
- Widely published and well-validated methodology
- Compatible with multiplexing (fluorescent detection)
- Only semi-quantitative without careful controls
- Low throughput (few samples per gel)
- Requires a validated antibody specific to the target
- Cannot provide spatial/localisation information
- Time-consuming (4–8 hours or overnight)
- Signal non-linearity limits absolute quantification
- Inter-experiment variation requires technical replication
Applications of Western Blotting
How western blot analysis is used in biomedical research
Western blotting applications span fundamental research, translational biology, and clinical diagnostics. Below are the most common use cases encountered in a research setting.
Signalling pathway analysis
Detect phosphorylated kinases or downstream effectors to map activation status under treatment conditions.
Protein expression validation
Confirm overexpression or knockdown after transfection, CRISPR editing, or siRNA treatment.
Disease biomarker detection
Detect aberrant protein levels or cleavage forms associated with cancer, neurodegeneration, or infection (e.g., HIV western blot confirmatory test).
Apoptosis & cell death
Detect cleaved caspases or PARP cleavage as markers of apoptotic signalling in treated cells.
Post-translational modification
Use phospho-, acetyl-, or ubiquitin-specific antibodies to determine when and how a protein is modified.
Co-IP and interaction studies
Western blotting used after immunoprecipitation to confirm that two proteins are in the same complex.
Frequently Asked Questions
Common questions about western blot analysis and protocol
- A standard western blot protocol takes 4–8 hours for the hands-on steps (sample prep, gel, transfer, blocking, antibody incubation). Primary antibody incubation is commonly performed overnight at 4°C, making the total elapsed time approximately 18–24 hours. Rapid protocols using semi-dry transfer and high-sensitivity detection reagents can compress total hands-on time to under 4 hours.
- Open your TIFF image in ImageJ. Use Analyze → Gels → Select First Lane to draw a rectangular ROI around the first lane, then advance through subsequent lanes with Select Next Lane. Select Plot Lanes to generate intensity profiles, then use the straight-line tool to close each peak baseline and the wand tool to measure peak area. Export values to Excel, subtract background, then divide each target band value by the corresponding loading control band value in the same lane to obtain a normalised density.
- Multiple bands can arise from: (1) antibody cross-reactivity with structurally similar proteins; (2) splice variants or isoforms of the same gene that differ in size; (3) post-translational modifications that shift a portion of the protein to a higher apparent mass; (4) proteolytic degradation generating a cleavage fragment; or (5) protein oligomerisation if samples are not fully denatured. Run a KO cell line to determine which bands are target-specific.
- SDS-PAGE (gel electrophoresis) separates proteins by size but visualises all proteins non-specifically using dyes such as Coomassie blue. Western blot (immunoblotting) goes further: after electrophoresis, proteins are transferred to a membrane and detected with a target-specific antibody, enabling identification of a single protein within a complex mixture.
- A loading control (e.g., beta-actin at ~42 kDa, GAPDH at ~37 kDa) should show equal band intensity across all lanes if the same amount of protein was loaded and transferred. If one lane shows a fainter loading control, that lane had less protein. Always normalise target band intensity to the loading control band in the same lane before drawing conclusions.
- Chemiluminescent detection (ECL) is more sensitive (down to picogram levels) and lower cost, but has a narrow linear range and is prone to saturation on film. Fluorescent detection offers a wider linear dynamic range (better for accurate quantification), enables multiplexing, and does not require a substrate step, but requires a fluorescent imaging system and is less sensitive than ECL at very low protein amounts.
- Western blot is inherently semi-quantitative; it measures relative changes between conditions rather than absolute protein concentrations. To approximate absolute quantification, you would need to run a standard curve of known amounts of recombinant protein in parallel and ensure your data is captured within the linear range. For true absolute quantification, techniques such as ELISA, mass spectrometry-based proteomics with isotope-labeled standards, or Simoa are more appropriate.
- Best practice: (1) show a representative blot image with clearly labeled lanes, size markers, and antibody target; (2) include the loading control blot below the target blot; (3) present quantification as a bar or dot plot showing normalised densitometry values from at least three independent biological replicates; (4) show individual data points; (5) state the normalisation method, software used, and statistical test in the methods section; (6) do not crop or adjust images in ways that misrepresent the data; (7) report the number of biological replicates (n) and define error bars (SD or SEM).
Next Steps and Related Resources
Dive deeper into individual western blotting stages
Each step in the western blotting workflow has its own optimisation considerations. Use the guides below to troubleshoot specific stages or explore adjacent techniques.