Techniques in Protein Biochemistry Flashcards
assay
determines whether the protein of interest is present
salting in
Most proteins require some salt to dissolve in water
salting out
As the salt concentration is increased, different proteins will precipitate at different salt concentrations
Gel filtration chromatography
allows the separation of proteins on the basis of size
A glass column is filled with porous beads
large
proteins cannot enter the beads and exit the column first
Small proteins can enter the beads and thus have a longer path and exit the column last
Ion exchange chromatography
allows separation of proteins on the basis of charge
The beads in the column are made so as to have a charge
When a mixture of proteins are passed through the column, proteins with
the same charge as on the column will exit the column quickly
Proteins are released from the ion-exchange support by:
1. Increasing the salt concentration
2. Adjusting the pH of the mobile phase
Ion-Exchange Types
- Cation-Exchange
* Carboxymethyl (CM)
* Sulfopropyl (SP) - Anion-Exchange
* Diethylamino (DE)
* Quaternary amine (Q)
Affinity chromatography
takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups
Affinity Chromatography Procedure:
1. Aproteinmixtureispassedthroughthecolumn
2. OnlyproteinwithaffinityfortheaQachedgroupwillberetained
3. Theboundproteinisthenreleasedbypassingasolu.onenrichedinthe chemical to which the protein is bound.
High-pressure liquid chromatography (HPLC)
uses very fine beads in metal columns and high-pressure pumps to move the liquid through the column
gel electrophoresis
Proteins will migrate in an electrical field because they are charged. When the migration occurs in a gel
dialysis
The salt can be removed from a protein solution by dialysis.
* The protein solu.on is placed in a cellophane bag (dialysis membrane)
* The dialysis membrane with pores too small to allow the protein to diffuse
* However, the pores are large enough to allow the salt to equilibrate with the solution surround the dialysis membrane
Native PAGE
- Proteins can be purified without the use of SDS and denaturing conditions
- Native PAGE is useful in determining the MW of the quaternary structure of the protein being analyzed
- Native PAGE is one-dimensional
isoelectric point
If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate to their isoelectric point, the pH at which they have no net charge.
