Biochemistry (Subject) / Proteins (Lesson)

This lesson was created by FriediFehlau.

Learn lesson

back  |  next 1 / 1

• cell disruption methods osmolytic repeated freezing/thawing shear forces(blending, homogenizer) ultrasound
• differential centrifugation steps in Potter-Elvejhem Homogenizer 1. low speed: 600g, 10'- nuclei, unbroken cells 2. higher speed: 15000g, 5'- mitochondria, lysosomes 3. ultra-speed: 100000g 60'- pellet: ribosomes, ER, Golgi, plasma membrane- supernatant: soluble fraction of cytoplasm
• Laemmli Buffer SDS: denaturationß-mercaptoethanol: breaking of disulfide bridgesTRIS/HCL buffer: glycine trailing ionGlycerol: high-density loading substanceBromphenol blue: tracking dye
• Western blot after sds, proteins are transferred o a membrane via volagemembrane is blocked with other proteins to prevent unspecific bindingFirst AB is added to the membrane to specifically detect the proteins of interestsecond AB is added with detection marker
• Biuret assay complex of Cu2+ ions with peptide bonds in alkaline solutiondisadvantage: low sensitivity, dependent on tyr content because it also forms a complex
• Lowry assay Reduction of Folin reagent catalysed by biuret assayadvantage: tolerant to low concentrations of SDS, increased sensitivity
• BCAssay protein Cu2+ complexes are reduced with bicinchonic acid (BCA)advantages: high sensitivity, tolerant to detergents, sensitivity can be optimised by temperaturedisadvantage: perturbed by reducing agents, expensive, contributions of side chains
• Bradford assay coomassie brilliant blue stains basic side chains -> absorbance from 465 to 595advantage: simple and quick, very sensitive, tolerates reducing agentsdisadvantage: small linear range, sensitive to sds and other detergents, highest subjectivity to sequence, as it mainly bind arginine -> standard should be similar to protein
• competitive inhibition substrate and inhibitor compete for active site,  same Vmax, increased Km
• non-competitive inhibition inhibitor binds to allosteric site, conformational change decreased Vmax, same Km
• Advertisement
• uncompetitive inhibition inhibitor combines with enzyme substrate complex Decrease of Vmax and Km
• Coenzymes organic molecules participating in a reaction e.g. biotin, CoA, nicotinamide coenzymes
• holoenzyme active enzyme-cofactor complex
• Apoenzyme enzymatically inactive protein lacking cofactors
• Enzyme classification 1. oxidoreductases 2. transferases 3. hydrolases 4. lyases 5. isomerases 6. ligases
• zymogen inactive precursor, activated by cleavage e.g chymotrypsin, activated by cleavage via trypsin
• abzymes Antibodies with catalytic activity
• ribozymes RNAs with catalytic potential

back  |  next 1 / 1