1. Biochemistry and molecular biology labLecture 18Serum protein separation bycellulose acetate membraneelectrophoresis

2. Biochemistry and molecular biology labAim Learn the principle of cellulose acetatemembrane electrophoresis Know the operation and clinical significance ofelectrophoresis 3. Biochemistry and molecular biology labPrinciple Electrophoresis:is the motion of charged particles relative to afluid under the influence of an electric field.Factors determining the electrophoresis motion charge, size, sharpBased on different supporting materials, there are membraneelectrophoresis and gel electrophoresis Gel electrophoresis is the process by which moleculesin a sample can be separated by charge and/or size. 4. Biochemistry and molecular biology labLab equipments electrophoresis deviceelectrophoresis chamber DYY-2 model power supplyThe connection between electrophoresis chamber and power supply 5. Biochemistry and molecular biology lab cellulose acetate membrane2 cm8 cm Petri dishstaining and rinsing Sample applicator (plastic slice) Filter paper Watch glass Forceps 6. Biochemistry and molecular biology lab Rocking table 722 modelspectrophotometer 7. Biochemistry and molecular biology labReagents fresh serum Barbitone sodium Buffer pH8.6 Staining solutionamido black 10B Destaining solution 0.4mol/L NaOH 8. Biochemistry and molecular biology labExperimental steps1. MembranepreparationSoaking the membrane withbuffer for more than 30mTinake the membrane with forceps, place themembrane between two pieces of filter paper todry the buffer (not too dry), and differentiatebetween the smooth and rough surface (roughsurface faces up).NoteOnly touch the margins of themembrane 9. Biochemistry and molecular biology lab2. Sample spottingTake a small amount of serum with the plastic slice, and stamp on the membraneNotesMark withpencil Spotting on the rough surface, 1.5cm from one membrane shortedge softly press for 1-2 sec, let the serum absorbed by the membrane Spotting only once, no need to repeat Spot requirements: thin, straight and not reaching the long edge 10. Biochemistry and molecular biology lab3. Place the sampleConnect the power supply with electrophoresis chamberLet the rough surface faces down, the spotting side isplaced at the cathode (-) side (Note: do not let the spotoverlap with the supporting paper)Remove bubbles between the membrane and supportingpaper 11. Biochemistry and molecular biology lab4. Electrophoresis Make sure that the membrane is wet Pre-electrophoresis50V, 5min ElectrophoresisStable voltage, 110V, 40 min 12. Biochemistry and molecular biology lab4. Staining and rinsing StainingTransfer the membrane using forceps to the Amido black 10Band stain for 3 minNoteOnly touch the margin regions of the membrane withforcepsCompletely submerge every membranes into the stainingsolution, no overlapping.Rinsingdetaining on the rocking table for 3 times(8 min; 7 min; 6 min)serum albumin globulins Starting point 13. Biochemistry and molecular biology lab5. QuantificationCut each bands and a part of the blank membrane, add 4mlNaOH respectively and shake for 15 min. Determine the lightabsorbance at 620 nm.6. CalculationTotal absorbance T= A +1+ 2+ +Percentage = ( X/ T ) 100%A/G = A/(1+ 2+ +) 14. Biochemistry and molecular biology labResultsserum albumin1 -globulins2 -globulins- globulins- globulins 15. Biochemistry and molecular biology labNormal range serum albumin 57.45-71.73% 1-globulins 1.76-4.48% 2-globulins 4.04-8.28% -globulins 6.79-11.39% -globulins 11.85-22.97% A/G 1.24-2.36 16. Biochemistry and molecular biology labClinical significance Most of serum protein is produced by liver only-globulins produced by plasma cells The function of serum proteins Maintain plasma colloid osmotic pressures; maintainplasm pH balance, base-acid balance; Transportnutrients, metabolites, hormones, medicines and metalions. 17. Biochemistry and molecular biology lab liver cirrhosisserum albumin, 1, 2 - globulins Hepatocarcinomabetween albumin and globulins,there is an alpha feto protein (AFP) band acute and chronic nephritis & nephrotic syndrome serum albumin 2 and globulins Multiple myeloma serum albumin - globulins between and - globulins, there is a M band 18. Biochemistry and molecular biology labAssignment questions1.Which side (anode or cathode) shall we placethe sample during electrophoresis? Why?2. What are the possible reasons causing theirregular, distorted or atypical electrophoresisbands 19. Biochemistry and molecular biology labOther electrophoresis techniques 1SDS-Polyacrylamide Gel Electrophoresisusually used for protein molecular weightdetermination 20. Biochemistry and molecular biology lab2Isoelectric focusing electrophoresisa technique for separating different molecules by differences intheir isoelectric point (pI)32D electrophoresisan important technique in the proteomics4Agarose gel electrophoresisDNA or RNA separation 21. Biochemistry and molecular biology labAgarose Gel ElectrophoresisGel electrophoresis is a widely used technique for theanalysis of nucleic acids and proteins. Agarose gelelectrophoresis is routinely used for the preparation andanalysis of DNA.Gel electrophoresis is a procedure that separates molecules on the basis oftheir rate of movement through a gel under the influence of an electrical field.We will be using agarose gel electrophoresis to determinethepresence and size of PCR products. 22. Biochemistry and molecular DNA is negatively charged. biology lab When placed in an electrical field, DNA will migrate toward the positivepole (anode). An agarose gel is used to slow the movement of DNA and separate bysize.- +PowerDNA Polymerized agarose is porous,allowing for the movement of DNAScanning Electron Micrographof Agarose Gel (11 m) 23. Biochemistry and molecular biology labHow fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gelSize of the DNA!*Small DNA move faster than large DNAgel electrophoresis separates DNA according to size- +PowerDNAsmalllargeWithin an agarose gel, linear DNA migrate inverselyproportional to the log10 of their molecular weight. 24. Biochemistry and molecular Agar boioslogey labD-galactose 3,6-anhydroL-galactoseSweetened agarose gels havebeen eaten in the Far East sincethe 17th century.Agarose was first used in biologywhen Robert Koch used it as aculture medium for Tuberculosisbacteria in 1882Agarose is a linear polymer extracted from seaweed. 25. Biochemistry and molecular biology labMaking an Agarose GelAn agarose gel is preparedby combining agarosepowder and a buffersolution.Buffer