7 Common Methods to Quantify Proteins – Pros and Cons

A side-by-side look at widely used techniques in protein analysis.

Protein quantification is a fundamental step in many workflows — from early discovery to therapeutic production. But no single method is universally ideal. This guide compares six commonly used techniques, each with its own strengths and trade-offs.

Note: Timings, performance, and workflows are based on typical lab protocols and may vary depending on the platform, reagents, and specific use case.

1. BCA Assay (Bicinchoninic Acid)

Principle: Proteins reduce Cu²⁺ to Cu⁺ under alkaline conditions; Cu⁺ forms a purple complex with BCA.
Pros:
✔ Detergent-tolerant
✔ Moderate dynamic range
Cons:
✘ Sensitive to reducing agents (e.g. DTT)
✘ Requires incubation time and heating in some protocols

2. Bradford Assay

Principle: Coomassie dye binds to basic and aromatic residues, shifting absorbance.
Pros:
✔ Fast and inexpensive
✔ Simple workflow
Cons:
✘ Sensitive to protein composition
✘ Narrow dynamic range

3. ELISA (Enzyme-Linked Immunosorbent Assay)

Principle: Antibody-based detection using enzymatic signal amplification.
Pros:
✔ High specificity
✔ Excellent for low-abundance proteins
Cons:
✘ Multi-step, time-consuming
✘ Requires validated antibodies
✘ Prone to inter-assay variability

4. Octet (Biolayer Interferometry / BLI)

Principle: Measures optical interference as proteins bind to biosensor tips.
Pros:
✔ Real-time monitoring
✔ Label-free
✔ Suitable for kinetics and quantification
Cons:
✘ Expensive instrumentation
✘ Requires well-controlled sample conditions
✘ Lower throughput than plate-based methods

5. SPR (Surface Plasmon Resonance)

Principle: Detects changes in refractive index at a sensor surface upon protein binding.
Pros:
✔ Label-free detection
✔ High sensitivity and kinetic data
Cons:
✘ Complex setup
✘ Costly and low throughput
✘ Sensitive to buffer mismatches

6. UV Absorbance (Nanodrop™ / A280 Measurement)

Principle: Measures absorbance at 280 nm, where aromatic amino acids (mainly tryptophan and tyrosine) absorb UV light.
Pros:
✔ Very fast — no reagents or incubation required
✔ No consumables beyond the sample itself
Cons:
✘ Assumes a known extinction coefficient
✘ Poor specificity — detects nucleic acids and other UV-absorbing components
✘ Not suitable for crude or unpurified samples
✘ Sensitive to buffer composition and baseline drift

7. RED (Redox Electrochemical Detection) — Used in Amperia™

Principle: Surface-bound proteins participate in an enzymatic redox reaction, generating an electrochemical signal proportional to protein concentration.

Pros:
✔ No optical components required
✔ Simple, software-guided workflow
✔ Compatible with crude and partially purified samples
✔ Flexible throughput — suitable for both small-scale and higher-throughput runs
✔ Validated for AAVs, monoclonal antibodies, and His-tagged proteins

Cons:
✘ As a newer technology, some users may need brief familiarisation compared to legacy methods