15 Biochemistry _ Experiment

• 1. Biochemistry and molecular biology labExperiment 4Folins phenol Method ToAnalyze Total Serum Protein

2. Biochemistry and molecular biology labClinical significanceThe reference range for total protein is typically 60-80g/L. (It isalso sometimes reported as "6.0-8.0g/dl"), but this may varydepending on the method of analysis.Concentrations below the reference range usually reflect lowalbumin concentration, for instance in liver disease or acuteinfection. Rarely, low total protein may be a signof immunodeficiency.Concentrations above the reference range are foundin paraproteinaemia, Hodgkin's lymphoma, leukaemia or anycondition causing an increase in immunoglobulins. Total proteinis also commonly elevated in dehydration. 3. Biochemistry and molecular biology labPurpose Learning Folins phenol method ofanalyzing serum protein Practice standard curve determination 4. Biochemistry and molecular biology labAmino acid residues form a complex withCu2+ ion in basic medium 5. Biochemistry and molecular biology lab FFoolliinnss pphheennooll mmeetthhooddThose protein containing phenol group can reduceSStteepp 11In alkalinecondition, Cu2+form complexwith proteinThe Cu-proteincomplex reducemolybdate.phosphotungstic acidand phosphomolybdate in blue color.SStteepp 22In blue color, the A value correlates with proteinconcentration at 660nm. 6. Biochemistry and molecular biology labMMiinniimmaall aannaallyyzziinngg ccoonncceennttrraattiioonn iiss 55 gg//mmll,,lliinneeaarr ddeetteeccttiioonn rraannggee iiss 2255--225500 gg//mmll .. AAddvvaannttaaggee:: DDiissaaddvvaannttaaggee::easy and reliable to operateProtein specificity can influencethe result; as well as otherinterfere substances. 7. Biochemistry and molecular biology labTTrriiss bbuuffffeerr gglluuccoosseeIInntteerrffeerreessuubbssttaanncceessEEGGTTAAAAmmmmoonniiaa ssuullffaatteePPhheennoolliicc ccoommppoouunnddssUUrreeaacciittrraatteegguuaanniiddiinneessooddiiuumm ssuullffaatteeTTCCAAaallccoohhoollaacceettoonneeCCHHAAPPSSEEDDTTAAHHEEPPEESSTTrriittoonn XX--110000ddiitthhiiootthhrreeiittooll 8. Biochemistry and molecular biology labThe reagent does not only measure phenols, and willreact with any reducing substance. It therefore measuresthe total reducing capacity of a sample, not just phenoliccompounds. This reagent is part of the Lowry proteinassay, and will also react with some nitrogen-containingcompounds such as hydroxylamine and guanidine.The reagent has also been shown to be reactivetowards thiols, many vitamins, the nucleotide baseguanine, the trioses glyceraldehyde anddihydroxyacetone, and some inorganic ions. Coppercomplex increases the reactivity of phenols towards thisreagent. 9. Biochemistry and molecular biology labApparatus and reagents Large tube, measuring pipette, water bath; 722E UV-vis spectrophotometer; Folin reagent (including reagent I and II); standard protein solution(250g/ml); serum sample (diluted 500 times); 10. Biochemistry and molecular biology labIn 7 large tube, Procedure adding reagents as following tableBlank(1) (2) (3) (4) (5) (6)Sample 7Standard protein solution(250g/ml) -- 0.2 0.4 0.6 0.8 1.0 Serum sample 1.0Distilled water 1.0 0.8 0.6 0.4 0.2 0 --Folin reagent I 3.0 3.0 3.0 3.0 3.0 3.0 3.0Mixture, and standing for 10min at room temperature.Folin reagent II 0.3 0.3 0.3 0.3 0.3 0.3 0.3Mix quickly !Standing for 30minDetecting absorption at 660nm, and obtain A2-A7. 11. Biochemistry and molecular biology lab Calculation and Drawing StandardCurve SSttaannddaarrdd CCuurrvvee MMeetthhoodd//CCaalliibbrraattiioonn CCuurrvvee SSttaannddaarrdd CCoonnttrrooll MMeetthhoodd 12. Biochemistry and molecular biology labIn alkaline solution, pyruvate-dinitro phenylhydrazones emerge reddish-brown color withthe absorption peak in 510nm. Theabsorbance value linearly correlates withpyruvate concentration. lambert-beer law: A= c lcsolution concentrationlliquid thicknessAsubstance absorbancemolar absorbance coefficient 13. Biochemistry and molecular biology labExperiment resultsStandard tube sample 14. Biochemistry and molecular biology labMODE bottomsetting detection mode;100%Tbottomsetting 100.0%Tautomatically;0%Tbottomsetting 0.000%Tautomatically;wavelengthsettingknob722S mode722E modeUse [MODE] button setting test manner: Transmittance (T), Absorbance (A),Concentration (C). 15. Biochemistry and molecular biology lab SSttaannddaarrdd CCuurrvveeA standard curve is a type of graph used as a quantitative research technique.Multiple samples with known concentrations are measured and graphed, whichthen allows the concentration of unknown samples to be determined byinterpolation on the graph. The absorbance value of each standard is thendetermined by spectrophotometer. Plot a standard curve with the knownconcentrations of protein, and the corresponding absorbance. The absorbancevalue of a solution of unknown concentration is then determined under identicalconditions and the concentration can be simply read from the plot. 16. Biochemistry and molecular biology labStandard curve CAA6A5A4A3A20 C2 C3 C4 C5 C6C7A7According to the standard plot, we can easily obtain C7 fromthe curve. 17. Biochemistry and molecular biology labCalculation of protein concentration for the sample(The serum has been diluted 500 fold)SSaammppllee pprrootteeiinnccoonncceennttrraattiioonn==CC77gg//LL 11000000550000 18. Biochemistry and molecular biology labtube 1 2 3 4 5 6 7OD660nm 0 0.121 0.250 0.335 0.440 0.525 0.320MXAXMM77gg//mmll55000011000000 CCsseerruumm==114499 ==gg//mmll7744..55gg//LL 19. Biochemistry and molecular biology labNotice at least 5 dot in a standard curve; standard solution and sample solution should be analyzed under thesame condition; the concentration of the solution should be in the linear range, andabsorbance value should be in the range of 0.05 1.0; 20. Biochemistry and molecular biology labStandard control methodCsample = Asample / Astandard Cstandard1. write all of those primary data;2. calculation;3. adding unit in concentration. 21. Biochemistry and molecular biology labTube 1 2 3 4 5 6 7OD660nm 0 0.121 0.250 0.335 0.440 0.525 0.320CCsseerruumm==00..33220000..33335500..2255VVssttaannddaarrddVVssaammppllee00..6611AAssaammpplleeAAssttaannddaarrddCCssttaannddaarrdd 550000== 7711..66 gg//LLNormal range of human serum protein is60-80g/L 22. Biochemistry and molecular biology labAssignment question What is standard curve