Protein Purification Techniques PDF

Part 1 : protein purification techniques 1 ). centrifugation force & density separate...

Part 1 : protein purification techniques 1 ). centrifugation force & density separate of substance through centrifugal # gravity ↳ the component ① Differential centrifugation ② Rate zonal => - centrifugation density gradient centrifugation ↳ Sucrose solutiona => same band means substance has same density => than the before according the weight every , centrifugation would be higher speed 1 2. Dialysis ↳ through the selectively membrane to separate molecules ↳ separation based on size ↳ diffusion down the conc. gradient 1 3. affinity chromatography ① only specific protein that bind to specific antibody in the first eluent, non-binding protein will be eluted out ② add pHt3 buffer (make protein protonated) ③ break down &S bond & non-covalent interaction > so can - go out How to remove molecule of interest : ① buffer with different ply/ion strength ② competitive inhibitor peptide tags - 14. exchange chromatography => separate molecules based on their electrical charge Damaged on => strength of interaction % protein molecule & ion exchange matrix depend St an ↳ charge density on the protein => second eluent ↳ You of mobile phase strength pH pl negative charge > : Canion) pH)pI positive charge = (cation)  chromatogram E 1. 5 size exclusion chromatography => separate molecules according to their size Step : ① put different size of molecule inside time ② smaller size molecules will be inside the beads trapped ③ larger molecule will move down first smaller molecule would be second eluent 1 6 1 Fast Protein liquid Chromatography (FPLC) => liquid chromatography => mixture of use to analyze/purity proteins => different columns has different separation resolution different , different size of A peak protein Part 2 : Protein detection & characterization 2 1. Polyacrylamide Gel Electrophoresis (PAGE) c a tea field Tot => molecules are separated by size in an electric croteins => it moves from ve to the => longer the peptide chain (move-re) fewer , the distance it moves Gel electrophoresis ① (SDS-PAGE) ↓ Denaturing charge T => SDS (detergant) gives uniform ve - => separtes protein by size/mass ② Non-denaturing based size/conformation => separate on charge & & determine of cannot 3D structure protein can show more band (heavy chain & light chain of human (g-G) => often combined with Western blotting different antibodies specific for proteins of interest) Cusing ① denaturation first , by mercaptoethanol ② add SDS > let protein bond to-re charged - SDS & break non-covalent bond of protein ③ undergoes electrophoesis again => fluorescent staining silver staining coomassive blue staining : , , staining.) Isoelectric 2 focusing => separate according to their pl(isoelectric points => different have different protein pl value zwitterion form E Inpl , they exist in (no net charge) => electric field , protein under the migrate to pl value · krp B B 2) gel electrophoresis 1) Ist dimension based ↳ separation on pl ↳isoelectric 2) 2nd dimension focusing of zwitterions ↳ normal SDS-PAGE ↳ ectrophe 23. Western blotting Step ① detect the antibody first bind to primary antibody , ② develop the antibody , bind to secondary antibody 2. 4 Edman Degradation O acid complete hydrolysis of peptide bonds ↳ determine acid amino composition ② Edman Degradation ↳ determine peptide of sequencing ③ larger proteins cleared into smaller are peptide by chemical/enzymatic cleavage Complete the protein sequence : > - find the amino acid & bind them  2 5 Mass. => determine spectrometry acid amino sequence Dionization ② acceleration ③ deflection ⑪ detection 2 6 chemical of. synthesis peptides E * acid -Butylocarbonyl amino Dicyclohexylcarbodiimide 27 Methods for. determining protein structure OX-ray crystal ographyw structure of protein & NMR (nuclear resonance) magnetic => analyze small/medium size of protein ③ Cryo-electron (cryo-EM) microscopy => the latest method => put protein in freezing & help it stabilizing in vaccum X-ray NMR cryo-EM CrystallographynoneedCrystal largely organelle as " need crystal only analyze small" low resolution medium of i need crystal protein