Size Exclusion Chromatography
larger moves faster, smaller moves slower
Ion Exchange Chromatography
oppositely charged beads stick to column, everything else flows through
Affinity Chromatography
Specific trap for protein of interest attached to stationary phase; high purity can be achieved
Order of specificity of different chromatography methods
- affinity
- ion exchange
- size exclusion
electrophoresis
migration of ions in an electric field
In electrophoresis, velocity is directly proportional to ______ and inversely proportional to_________.
charge, size and shape
A gel is used to…
slow down movement, prevent diffusion of proteins, and create separation
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDS
a detergent that will denature proteins and give them a negative charge
How much SDS for 1g protein?
~1.4g
acrylamide
acrylic amide; a plastic monomer used industrially that can be chained together
monomers
toxic, carcinogenic and teratogenic, but safe when linked to gel
SDS-PAGE procedure
- protein samples boiled for 5mins in 5xloading buffer
- gel is cast by mixing acrylamide, bisacrylamide, APS, TEMED, SDS, and buffer
- gel placed vertically in electrophoresis apparatus, tanks are filled with buffer
- samples loaded into wells (ladder important to determine sizes)
- power turned on until blue dye reaches bottom
Visualizing gel
-coomassie blue, silver nitrate (complicated and time consuming), fluorescent dye (require specialized equipment)
