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Protein methods 2010 lab 3 protein extraction
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.
Contents
- Protein methods 2010 lab 3 protein extraction
- Protein methods 2010 lab 4 affinity chromatography
- Genetic methods
- Protein extraction from tissues
- Protein purification
- Non specific methods that detect total protein only
- Specific methods which can detect amount of a single protein
- Protein structures
- Interactions involving proteins
- Proteinprotein interactions
- ProteinDNA interactions
- ProteinRNA interactions
- Computational methods
- Other methods
- References
Protein methods 2010 lab 4 affinity chromatography
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. Some examples of genetic methods include conceptual translation, Site-directed mutagenesis, using a fusion protein, and matching allele with disease states. Some proteins have never been directly sequenced, however by translating codons from known mRNA sequences into amino acids by a method known as conceptual translation. (See genetic code.) Site-directed mutagenesis selectively introduces mutations that change the structure of a protein. The function of parts of proteins can be better understood by studying the change in phenotype as a result of this change. Fusion proteins are made by inserting protein tags, such as the His-tag, to produce a modified protein that is easier to track. An example of this would be GFP-Snf2H which consists of a protein bound to a green fluorescent protein to form a hybrid protein. By analyzing DNA alleles can be identified as being associated with disease states, such as in 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.