Protein quantification is a fundamental step across many laboratory workflows, from early discovery through to therapeutic production. With so many protein quantification methods available, choosing the right one depends on your sample, your target, and what the data will be used for.
This guide compares seven common methods to quantify proteins, explains when each is most appropriate, and highlights the practical considerations that matter when choosing a method for your workflow.
Timings and workflow details are based on typical laboratory protocols. They may vary depending on the platform, reagents and specific application.
BCA Assay (Bicinchoninic Acid)
BCA is a colorimetric assay based on the reduction of Cu²⁺ to Cu⁺ by proteins in alkaline conditions. The resulting Cu⁺ ions form a purple complex with bicinchoninic acid, producing a signal proportional to protein concentration.
When to use it: BCA is a good general-purpose choice when a straightforward total protein measurement is required from relatively clean samples. It is commonly used for lysate characterisation, protein normalisation and basic yield checks.
Typical workflow: Add reagent to sample, incubate at 37°C for 30 minutes, read absorbance at 562 nm.
Pros:
- Detergent-tolerant, compatible with many common lysis buffers
- Moderate dynamic range (around 20 to 2,000 µg/mL)
- Well-established with widely available commercial kits
Cons:
- Sensitive to reducing agents such as DTT and β-mercaptoethanol
- Requires incubation time
- Measures total protein, cannot distinguish a target from contaminants
Bradford Assay
Bradford relies on Coomassie Brilliant Blue G-250 dye, which binds to basic and aromatic amino acid residues and shifts its absorbance maximum from 465 nm to 595 nm. Colour change intensity is proportional to protein concentration.
When to use it: Bradford suits situations where speed and simplicity are the priority and samples are relatively pure. It is fast, inexpensive and requires only a spectrophotometer or plate reader.
Typical workflow: Add Bradford reagent to sample, wait 5 minutes at room temperature, read absorbance at 595 nm.
Pros:
- Results in under 10 minutes
- Inexpensive and simple, single-step protocol
Cons:
- Response depends on amino acid composition, so BSA standards may not reflect the target protein accurately
- Narrow dynamic range
- Incompatible with most detergents (SDS, Triton X-100)
- Measures total protein only
Lowry Assay
The Lowry assay is a two-step reaction. Proteins first react with copper in alkaline solution, then the copper-protein complex reduces Folin-Ciocalteu reagent to produce a blue colour measured at 750 nm.
When to use it: Lowry has largely been superseded by BCA in most modern laboratories. It remains relevant for legacy protocols and when comparing against historical datasets.
Typical workflow: Add alkaline copper reagent, incubate 10 minutes, add Folin reagent, incubate 30 minutes, read absorbance at 750 nm.
Pros:
- Moderate sensitivity
- Well-characterised with decades of published data
Cons:
- Multi-step and hands-on
- Sensitive to many common buffer components including EDTA, carbohydrates, lipids and reducing agents
- Timing-sensitive, colour development must be read within a specific window
UV Absorbance (A280)
Aromatic amino acids, primarily tryptophan and tyrosine, absorb ultraviolet light at 280 nm. The absorbance is proportional to protein concentration when the extinction coefficient is known.
When to use it: UV absorbance is most suited to quick concentration checks on purified samples, where the priority is a quick estimate rather than precision. It is commonly used to monitor column fractions during purification or to check stock concentrations.
Typical workflow: Pipette 1 to 2 µL onto a UV-Vis spectrophotometer, read absorbance at 280 nm, calculate concentration using the Beer-Lambert law.
Pros:
- Results in seconds, no reagents or incubation required
- Non-destructive in some configurations
Cons:
- Requires a known extinction coefficient
- Nucleic acids, free amino acids and other UV-absorbing components interfere
- Not suitable for crude or complex samples
- Limited sensitivity for dilute samples
ELISA (Enzyme-Linked Immunosorbent Assay)
ELISA uses antibodies to bind the target protein. A capture antibody immobilises the analyte, a detection antibody recognises a second epitope, and an enzyme-linked conjugate produces a measurable colorimetric, fluorescent or chemiluminescent signal.
When to use it: ELISA is the standard choice when target-specific quantification is needed, particularly for measuring a single protein in complex samples such as cell culture supernatant, serum or lysate. It is widely used for antibody titre measurement, biomarker quantification and quality control. For more on the practical considerations of manual ELISA in bioprocessing workflows, see The Hidden Costs of Manual ELISA.
Typical workflow: Coat plate, block, add sample, wash, add detection antibody, wash, add substrate, read. A full plate typically requires several hours of hands-on and waiting time.
Pros:
- High specificity, measures the target rather than total protein
- Sensitive, can detect pg/mL concentrations
- Well-validated with extensive published protocols
Cons:
- Labour-intensive, multi-step protocol
- Requires validated, matched antibody pairs
- Prone to inter-assay, inter-operator and inter-plate variability
- Significant turnaround time per plate
BLI (Biolayer Interferometry)
BLI directs white light down a fibre-optic biosensor tip. Proteins binding to the tip surface create an interference pattern that shifts proportionally to the amount of bound protein, enabling real-time, label-free measurement.
When to use it: BLI suits laboratories that need both quantification and kinetic binding data from the same platform. It is commonly used in antibody characterisation, clone screening and process development where binding behaviour and concentration are both of interest.
Typical workflow: Hydrate sensors, load capture molecule, dip into sample, monitor binding curve in real time.
Pros:
- Real-time, label-free monitoring
- Provides quantification and kinetic data from the same run
- No wash steps required
Cons:
- Expensive instrumentation
- Sensor tips are single-use consumables with significant per-test cost
- Lower throughput than plate-based methods for pure quantification
- Sensitive to sample viscosity, bubbles and matrix effects
RED (Redox Electrochemical Detection)
RED uses functionalised sensor surfaces that capture a target protein and participate in a surface-coupled enzymatic reaction. The reaction generates an electrochemical signal proportional to protein concentration, measured without optical components. RED is the detection principle used in the Amperia™ system.
When to use it: RED is suited to workflows where target-specific measurement is needed alongside minimal hands-on steps, particularly when working with crude or partially purified samples where optical interference can be a challenge.
Typical workflow: Prepare samples in a 96-well plate, load sensor strips, start the software-guided run.
Pros:
- No optical components, less sensitive to sample turbidity or colour
- Compatible with crude and partially purified samples
- Software-guided workflow with minimal hands-on steps
- Flexible throughput, suitable for small runs or larger sample sets
Cons:
- Newer technology with a smaller published evidence base than ELISA
- Currently limited to specific analyte types including antibodies, AAV capsids and His-tagged proteins
- Requires the Amperia system
Protein quantification methods at a glance
| Method | Speed | Sensitivity | Specificity | Sample Prep | Instrumentation |
|---|---|---|---|---|---|
| BCA | Moderate | Moderate | Low | Moderate | Spectrophotometer |
| Bradford | Fast | Low–Moderate | Low | Low | Spectrophotometer |
| ELISA | Slow | High | High | High | Plate reader + antibodies |
| Octet (BLI) | Moderate | Moderate–High | Moderate–High | Moderate | Octet instrument |
| SPR | Moderate | High | High | High | SPR platform |
| UV Absorbance | Very fast | Low | Low | High purity | Nanodrop™ or UV-Vis spectrometer |
| RED (Amperia™) | Moderate | Moderate–High | Application-specific | Low–Moderate | Amperia™ system |
How to choose the right protein quantification method
The choice depends on three things: what you need to measure (total protein or a specific target), what your sample looks like (purified or crude) and what the data will be used for (screening or formal characterisation).
For a total protein estimate from a purified sample, UV absorbance or Bradford will give a result quickly. For target-specific measurement from a complex sample, the options are ELISA, BLI or electrochemical detection, each with different trade-offs around throughput, cost and hands-on time.
Among protein concentration methods aimed at specific targets, the right choice often comes down to practical fit with the sample type and the frequency of measurements, rather than raw sensitivity alone.
Frequently asked questions
What is the most accurate protein quantification method?
Accuracy depends on the sample and application. For target-specific measurement, ELISA is the most widely validated method, though it requires careful antibody selection and protocol optimisation. For total protein, BCA offers a good balance of accuracy and practicality for most sample types.
Can I quantify proteins in crude samples?
Total protein assays such as BCA, Bradford, Lowry and UV will measure everything in the sample, target and contaminants together. For crude samples, a target-specific method is required, typically ELISA, BLI or RED.
What is the difference between total protein and target-specific quantification?
Total protein methods (BCA, Bradford, Lowry, UV) measure all proteins in a sample combined. Target-specific methods (ELISA, BLI, RED) use antibodies or affinity reagents to measure only the protein of interest. Total protein methods are useful for general characterisation. Target-specific methods are required when the result needs to reflect a particular molecule rather than the whole mixture.
How do I choose between BCA and Bradford?
If your buffer contains detergents, BCA is detergent-tolerant. If it contains reducing agents such as DTT, Bradford is unaffected by them but is incompatible with detergents. If neither is a concern, Bradford is faster.
Are the same methods used for research and regulated workflows?
The underlying methods are often the same, but regulated environments typically require validated protocols, qualified standards and documented traceability. A method that is straightforward for research use may require additional qualification before it can be used in a GMP or clinical context.
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