Studying proteins and peptides in the lab
Purification, separation, identification, structure determination and peptide synthesis.
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- Match common lab methods to the question they answer
- Explain how SDS-PAGE and chromatography separate proteins
- Describe what mass spectrometry and structural methods reveal
- Outline solid-phase peptide synthesis
Key concepts
Separating and purifying
- Chromatography separates by size (size exclusion), charge (ion exchange), specific binding (affinity) or hydrophobicity (reverse-phase HPLC). See Understanding HPLC.
- SDS-PAGE: SDS unfolds proteins and coats them with negative charge, so they separate by approximate mass.
- Isoelectric focusing separates by pI; combined with SDS-PAGE it forms a 2D gel.
Identifying and sequencing
- Mass spectrometry (ESI or MALDI) measures mass very precisely and, in tandem mode, reads sequence. It is the standard way to confirm a peptide’s identity.
- Edman degradation removes and identifies one amino acid at a time from the N-terminus.
Seeing structure
X-ray crystallography, NMR spectroscopy and cryo-electron microscopy determine 3D structures, deposited in the Protein Data Bank. AlphaFold predicts structures from sequence.
Making peptides
Solid-phase peptide synthesis (Merrifield, Nobel Prize 1984) anchors the first amino acid to a resin and adds protected amino acids one at a time, building the chain from the C-terminus to the N-terminus.
Measuring how much
Absorbance at 280 nm (from Trp and Tyr), or colorimetric assays such as Bradford and BCA. Purity, identity, sterility and endotoxin levels are separate questions: see this guide.
Worked example
Problem: A peptide has 2 Trp and 1 Tyr. Its absorbance at 280 nm in a 1 cm cuvette is 0.25. What is its concentration?
Solution: ε = 2 × 5,500 + 1 × 1,490 = 12,490 M⁻¹cm⁻¹. By Beer–Lambert, c = A ÷ (ε × l) = 0.25 ÷ 12,490 = 2.0 × 10⁻⁵ M = 20 µM.
Common mistakes
- Treating an HPLC purity percentage as proof of identity. Identity needs mass spectrometry or another method.
- Treating SDS-PAGE mass as exact. It is an estimate.
- Trying to measure peptides without Trp or Tyr at 280 nm.
Check yourself
What property does SDS-PAGE separate proteins by?
In which direction is a peptide built in solid-phase synthesis?
Which technique best confirms a peptide’s identity?
17 cards on lab methods.
Go deeper
VALREXA Biosciences. (2026, November 7). Studying proteins and peptides in the lab: study guide. VALREXA Biosciences. https://valrexa.com/study/lab-methods.html
APA style · Last reviewed November 7, 2026 · Our editorial standardsYour next step
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