BIOCHEMISTRY EXPERIMENT Principle + Practice Logic analysis Professor: Sheng Zhao (赵晟) Web for lecture slides： http://teaching.ewindup.info Email/QQ /MSN/gchat: [email protected] or [email protected] Mobile:18551669724 or 18761413925 Class syllabus by logic Protein quantification: Folin phenol (Lowry) assay Ion exchange chromatography for the mixed amino acids Protein analysis Transamination Protein purification Gel filtration chromatography Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) Lipid analysis Nucleic acid analysis Serum triglyceride (TG) measurement Plasmid DNA extraction, restriction enzyme digestion, and agarose gel electrophoresis Enzyme Km Protein function Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!) Class syllabus by real experiments 1. Introduction and Protein quantification: Folin phenol (Lowry) assay 2. Protein purification: Gel filtration chromatography 3. Plasmid DNA extraction, restriction enzyme digestion, and agarose gel electrophoresis 4. Enzyme Km 5. Transamination 6. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDSPAGE) 7. Ion exchange chromatography for the mixed amino acids 8. Serum triglyceride (TG) measurement 9. Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!) Experiment: isolation of hemoglobin using the gel filtration chromatography Charge, size, solubility, and Specific binding, etc. Column chromatography • Charge • Ion-exchange chromatography: • Isoelectric focusing chromatography • Size • Size-exclusion chromatography • Solubility • Hydrophobic interaction chromatography • Specific binding • Affinity chromatography Column chromatography Size-exclusion Chromatography This method separates proteins according to their size. The column contains a cross-linked polymer with pores of selected size. Size Exclusion Chromatography: Applications of gel filtration • Purification of enzymes and other proteins. • Estimation of molecular weight mainly for globular proteins • Desalting • The method of gel filtration chromatography can be used to separate hemoglobin (Hb) from riboflavin according to their molecular weight. • Hb (MW: 64500 ): • Riboflavin (MW: 376) Regents: 1. Sephadex G-50; 2. 0.9 % NaCl 3. Non-coagulating blood 4. Riboflavin G50 Equipment and apparatus: 1. Chromatography column (diameter, 0.8-1.5 cm; length, 17-20 cm); 2. Bracket for chromatography column; 3. Centrifuge tube, burette, centrifuge. 1. Column filling 1. Sephadex G-50 will be used for the column filling 2. While filling the column, please notice that the chromatography column fixed on the bracket should be upright vertically. 3. Close the clamp for exit at the bottom, and add gradually and gently the sephadex G-50 suspending solution from the top of column. 4. When approximately 1-2 cm volume of sephadex G-50 is already deposited in the column, open the clamp for exit and the outflow speed should be adjusted as 1 ml/min. 5. Continue to finish the filling sephadex G-50 suspending solution gradually without drying the column. NOTICE: 1. NO bubble and layered deposition 2. Flat column surface (or the formed color band will be irregular) 3. Tightly closed clamp for exit 4. Never dry the column, and approximately 0.5 cm elution solution should be retained on the top of the surface of sephadex G-50 resin. 2. Sample preparation for hemoglobin 1. Add 1 ml blood into the centrifuge tube, balanced, and then centrifuge for 5 min at 3000 rpm. 2. Discard the supernatant (blood plasma), add 10 ml 0.9% NaCl, and mix thoroughly of the NaCl with blood corpuscle. Centrifuge again as above 3. Discard the supernatant and add 10 ml 0.9% NaCl again to wash the blood corpuscle for one more time. 4. And then, dilute the blood corpuscle with five volume of ddH2O (Why?). 5. Before the gel filtration, mix three drops of blood preparation with three drops of Riboflavin solution. Preparation of hb from blood: 3. Samples loading 1. Slightly open the clamp for exit to make the solution in column outflow slowly till the surface of sephadex G-50 resin just appears from the solution, then close tightly the clamp for exit. Do not make the surface of resin become dry, or your experiment will be failed. 2. Add the mix sample with burette on the surface of sephadex G-50 resin gradually along the inner wall, and do not destroy the surface of sephadex G-50 resin. 3. Open the exit to make the sample run into the stationary phase. While the surface of sephadex G-50 resin appears again, add quickly the 0.9% NaCl to elute the sample with the elution speed of 1ml/min. 4. Observe the elution and separation of Hb and riboflavin. 4. Recording the data • Record the elution sequence of the color. • Notice: the band color for hemoglobin is red, and the band color for riboflavin is yellow.