4 September, 2006

Chapters 20 - 21

Methods: Proteins,

Model Systems I

Overview• Different Protein molecules have different biophysical properties, so

methodology must be flexible and specific.• Protein purification requires a unique assay. • Column chromatography, including affinity methods, can be tailored to

the purification of proteins.• Denaturing (SDS) gels, combined with staining or immune detection are

used to separate and visualize proteins.• Protein molecules can be sequenced, but this is inefficient.• Virus are important model systems for studying DNA replication,

recombination and gene expression.• Bacterial systems are useful for studying molecular processes in a

system that is genetically tractable and readily cultured.• Bacterial and phage systems are the source of most fundamental

molecular biological knowledge.

Protein PurificationSpecific proteins can be purified from cell extracts, but this requires extreme care, attention to detail, and a specific assay.

Cells are lysed under controlled conditions, and proteins are separated by chromatography.

Affinity Purification

Knowledge about the molecules that bind a protein can be extremely useful in its purification.

Immunoaffinity chromatography is a useful variant of this technique.

Modern methods often modify proteins with tags or epitopes in expression vectors to facilitate their purification and detection.

Protein Gel Electrophoresis

Because proteins are diverse and have secondary structure, they are denatured and coated with SDS to give a constant mass:charge ratio before being separated by size.

Once separated, proteins can be detected by staining, autoradiography, or immunological methods

Purified proteins can be sequences using Edman Degradation or tandem Mass Spec.

Model Systems• Phage / Virus: DNA replication, Recombination, Regulation

• Bacteria: Minimal Cell

• S. cerevisiae: Minimal Eukaryote

• C. elegans: Development

• Drosophila: Genetics

• Mouse: Mammal

Phage

Bacteria: Minimal Cells• Single chromosome, rapid asexual reproduction.

• Haploid

• Prokaryotic

• Mobile genetic elements– Transduction– Transformation– Conjugation

• High mutation rate per locus

• Transposon Mutagenesis

Transposons

Bacteria Good