Biochemistry Protein Purification Lab Techniques
CHASM: Charge, Hydrophobicity or solubility, Affinity, Stability, and Molecular weight or size.
C- charge – Ion Exchange Chromatography
Cation and Anion Exchange: charged beads get attached to the proteins of the right charge
H: Salting out: Hydrophobicity
When you add salt such as NaCl to a solution of protein and water, eventually protein will precipitate out. The more hydrophobic protein the less salt it needs to precipitate out. Why? Salt ions will interact with water freeing proteins and making them come out
A-affinity- Affinity Chromotography
The columns is packed with beads. To these beads we attach specific group for the protein we’re trying to catch.
S- stability, for example heat
M – molecular weight
Gel Filtration Chromatography:
Separation of proteins based on their size, large proteins come out first. Small get stuck in the beads and take longer to come out.
Isoelectric Focusing: Separates proteins based on their pI. A gel with a pH gradient is made and each protein will move through the pressure of electric field until reaching PI.
SDS -Gel Electrophoresis:
Separates proteins by size, larger will move slower and stay at the top
2D Gel Electrophoresis: combines Isoelectric Focusing with SDS gel, so horizontally they get separated by PI and vertically by size
Antibodies: bind to antigens through the epitopic regions
Polyclonal: bind to different regions of the same antigen.
To get polyclonal antibodies we can: inject mouse with antigen and wait for it to make antibodies, draw blood, and isolate/purify antibodies.
Monoclonal antibodies: bind the same epitope of a specific antigen, arise from the same plasma cell.
Elisa- method that used antibodies to detect and quantify the presence of specific proteins.
Indirect Elisa- used to detect specific antibody.
Coat the surface of well with specific antigen, pour antibodies, and if its right it will bind to the antigen, enzyme linked antibodies binding to the antibodies of interest are added. Everything unbound is removed by washing. Substrate specific to enzyme is added, and reaction takes place changing the color of solution.
Detects the presence of antigen
Specific antibodies are attached to the well, antigens are poured and will attach if they are correct. Enzyme linked monoclonal antibody is added, then substrate à change of color.