Protein Analysis Techniques SDSPAGE Western Blotting MALDI SELDI
Protein Analysis
Techniques • • SDS-PAGE Western Blotting MALDI SELDI ELISA X-ray crystallography NMR
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
Western Blotting • Similar to northern and southern blotting but analyzes proteins instead of nucleic acids • Uses modified antibodies as probes – Enzyme-linked
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
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
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
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
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