Biochem Tests/chromatography

Front 1

• Chromatography

Back 1

Separation based on affinity for mobile phase versus stationary phase

• Size exclusion
o Separation based on size
o Larger proteins elute first

Front 2

Ion Exchange

Back 2

o Separation based on charge
o Anion exchange
• Anions bind + charges
o Cation exchange
• Cations bind – charges
o Found in water filters

Front 3

Hydrophobic interaction

Back 3

o Separation based on hydrophobicity of protein

Front 4

Affinity Chromatography

Back 4

o Stationary phase contains specific ligand
o Protein binds ligand, adheres to stationary phase
o Elute with excess ligand

Front 5

High Pressure Liquid Chromatography

Back 5

o Eluent pumped under high pressure
o Usually hydrophobic interaction
o Faster, higher resolution

Front 6

Native Electrophoresis

Back 6

• Separation based on native charge of proteins
• LDH enzymes

Front 7

SDS-Page

Back 7

• Separation based on protein size
• Single polypeptides separated, not whole proteins

Front 8

Isoelectric Focusing (IEF)

Back 8

• Proteins migrate to their pI values

Front 9

Two dimensional Gel Electrophoresis

Back 9

• IEF in the first dimension
• Do IEF first on strip then do SDS-page gel
• SDS-Page in the second dimension

Front 10

Western Blot

Back 10

• Separated proteins incubated with antibody
• Antibody-antigen reaction visualized

Front 11

Edman Sequencing

Back 11

o Chemical procedure removes an amino acyl residue from N-terminus
o Amino acyl residue may be identified
o Amin acyl residue sequence may be determined

Front 12

Mass Spectrometry

Back 12

o Molecules separated based on mass
o Proteins digested by trypsin
o Digested material subjected to mass spectrometry
o Proteins identified from their peptides identified by MS