Protein Analysis

1. Protein Analysis

2. Techniques • SDS-PAGE • Western Blotting • MALDI • SELDI • ELISA • X-ray crystallography • NMR

3. SDS-PAGE • Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis • SDS • Detergent • Denatures proteins; native conformations disappear and only primary structures are retained • PAGE • Uses polyacrylamide instead of agarose • Separation of protein fragments according to size

4. Western Blotting • Similar to northern and southern blotting but analyzes proteins instead of nucleic acids • Uses modified antibodies as probes • Enzyme-linked

5. MALDI and SELDI • Mass spectrometry • Matrix–Assisted Laser Desorption–Ionization • Proteins are immobilized in a matrix • Surface–Enhanced Laser Desorption–Ionization • Modification of the MALDI • Simpler steps for preparation

6. ELISA • Enzyme-Linked Immunosorbent Assay • Qualitative or quantitative protein analysis • Two types of antibodies are used • Primary binds to specific antigen • Secondary binds to antigen-primary antibody complex • Enzyme-linked; produces colorimetric reaction in the presence of substrate

7. X-ray crystallography • The ultimate technique in determining protein structure • A protein crystal is created then bombarded with an X-ray beam to create a diffraction pattern • The electron cloud and correspondingly, the structure, of the protein is calculated from the generated diffraction pattern

8. Nuclear Magnetic Resonance • NMR utilizes differences in the spin states of the nuclei of atoms found in the sample • Protein NMR is very complicated and can be used to elucidate the atomic backbone of the protein. • Utilizes computer algorithms to generate a partial 3-D structure of the protein