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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.
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.
Advantages of these devices include high levels of protein extraction from small, valuable samples, disadvantages include low-level cross-over contamination.
- Protein isolation
- Protein extraction and solubilization
- Protein concentration determination methods
- Concentrating protein solutions
- Gel electrophoresis
- Gel electrophoresis under denaturing conditions
- Gel electrophoresis under non-denaturing conditions
- 2D gel electrophoresis
- Microscopy and protein immunostaining
- Protein immunoprecipitation: technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
- Immunoelectrophoresis: separation and characterization of proteins based on electrophoresis and reaction with antibodies.
- Western blot: couples gel electrophoresis and incubation with antibodies to detect specific proteins in a sample of tissue homogenate or extract (a type of Immunoelectrophoresis technique).
- BCA assay (to quantify protein concentrations)
- Enzyme assay
Interactions involving proteins
- (Yeast) two-hybrid system
- Protein-fragment complementation assay
- Affinity purification and mass spectrometry
- Molecular dynamics
- Protein structure prediction
- Protein sequence alignment (sequence comparison, including BLAST)
- Protein structural alignment
- Protein ontology (see gene ontology)
- Hydrogen–deuterium exchange
- Mass spectrometry
- Protein sequencing
- Protein synthesis
- Peptide mass fingerprinting
- Ligand binding assay
- Eastern blotting
- Metabolic labeling
- Heavy isotope labeling
- Radioactive isotope labeling
- Daniel M. Bollag, Michael D. Rozycki and Stuart J. Edelstein. (1996.) Protein Methods, 2 ed., Wiley Publishers. ISBN 0-471-11837-0.