Protein methods

For methods with DNA or RNA, see Nucleic acid methods.

Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins, often requiring that the protein first be purified). Computational methods typically use computer programs to analyze proteins. However, many experimental methods (e.g., mass spectrometry) require computational analysis of the raw data.

Genetic methods

Experimental analysis of proteins typically requires expression and purification of proteins. Expression is achieved by manipulating DNA that encodes the protein(s) of interest. Hence, protein analysis usually requires DNA methods, especially cloning. Other examples include:

• Conceptual translation: many proteins are never directly sequenced, but their sequence of amino acids is known by "conceptual translation" of a known mRNA sequence. (See genetic code.)
• Site-directed mutagenesis allows new variants of proteins to be produced and tested for how structural changes alter protein function.
• Insertion of protein tags such as the His-tag. (See also green fluorescent protein.)
• Proteins that are involved in human diseases can be identified by matching alleles to disease and other phenotypes using methods such as calculation of LOD scores.

Protein extraction from tissues

Protein extraction from tissues with tough extracellular matrices (e.g., biopsy samples, venous tissues, cartilage, skin) is often achieved in a laboratory setting by impact pulverization in liquid nitrogen. Samples are frozen in liquid nitrogen and subsequently subjected to impact or mechanical grinding. As water in the samples becomes very brittle at these temperature, the samples are often reduced to a collection of fine fragments, which can then be dissolved for protein extraction. Stainless steel devices known as tissue pulverizers are sometimes used for this purpose (e.g., protein extraction tissue pulverizer).

Advantages of these devices include high levels of protein extraction from small, valuable samples, disadvantages include low-level cross-over contamination.

Detecting proteins

• Microscopy and protein immunostaining
• Protein immunoprecipitation
• Immunoelectrophoresis
• Immunoblotting
• BCA assay (to quantify protein concentrations)
• Western blot
• Spectrophotometry
• Enzyme assay

Protein purification

• Protein isolation
  • Chromatography methods: ion exchange, size-exclusion chromatography or Gelfiltration, affinity chromatography
• Protein extraction and solubilization
• Protein concentration determination methods
  • Bradford protein assay
• Concentrating protein solutions
• Gel electrophoresis
  • Gel electrophoresis under denaturing conditions
  • Gel electrophoresis under non-denaturing conditions
  • 2D gel electrophoresis
• Electrofocusing

Protein structures

• X-ray crystallography
• Protein NMR
• Cryo-electron microscopy

Interactions involving proteins

• Protein footprinting

Protein–protein interactions

• (Yeast) two-hybrid system
• Protein-fragment complementation assay
• Co-immunoprecipitation
• Affinity purification and mass spectrometry

Protein–DNA interactions

• ChIP-on-chip
• Chip-sequencing
• DamID
• Microscale thermophoresis

Computational methods

• Molecular dynamics
• Protein structure prediction
• Protein sequence alignment (sequence comparison, including BLAST)
• Protein structural alignment
• Protein ontology (see gene ontology)

Other methods

• Hydrogen–deuterium exchange
• Mass spectrometry
• Protein sequencing
• Protein synthesis
• Proteomics
• Peptide mass fingerprinting
• Ligand binding assay
• Eastern blotting
• Metabolic labeling
  • Heavy isotope labeling
  • Radioactive isotope labeling

See also

• CSH Protocols
• Current Protocols

Bibliography

• Daniel M. Bollag, Michael D. Rozycki and Stuart J. Edelstein. (1996.) Protein Methods, 2 ed., Wiley Publishers. ISBN 0-471-11837-0.