扬州大学：《生物化学 Biochemistry》课程教学课件（讲稿）Experiments for Bichemistry 2_Experiment 1and2

Experiment1:Determination of Protein ConcentrationUsing Coomassie Brilliant Blue StainingMethod

Experiment 1: Determination of Protein Concentration Using Coomassie Brilliant Blue Staining Method

l.ObjectivesandRequirements1.1 Learn and master the principles and operational methodsof the CoomassieBrilliant Blue staining method forproteinquantification.1.2 Further familiarize with the usage of the spectrophotometer

1. Objectives and Requirements 1.1 Learn and master the principles and operational methods of the Coomassie Brilliant Blue staining method for protein quantification. 1.2 Further familiarize with the usage of the spectrophotometer

2.FundamentalPrinciples The Coomassie Brilliant Blue staining method, establishedby Bradford in 1976 (also known as theBradford method)is based on the quantitative binding of proteins withCoomassie Brilliant Blue G-250When Coomassie Brilliant Blue G-250 binds to proteins, itsmaximumabsorptionpeak shiftsfrom465nmto595nm

2. Fundamental Principles  The Coomassie Brilliant Blue staining method, established by Bradford in 1976 (also known as the Bradford method), is based on the quantitative binding of proteins with Coomassie Brilliant Blue G-250.  When Coomassie Brilliant Blue G-250 binds to proteins, its maximum absorption peak shifts from 465 nm to 595 nm

2.FundamentalPrinciplesUnder conditions where Coomassie Brilliant Blue G-250 ispresent in excess and at a constant concentration, varyingprotein concentrations in the solution result in differentamounts of dye molecules transitioning from the 465 nmabsorptionformtothe595nmform.This transition follows a quantitative relationshipGenerally,asproteinconcentrationincreases,theabsorbanceof the reagent at 595nm increases nearlylinearly, allowing the Coomassie Brilliant Blue G-250stainingmethodto be usedforproteinquantitation

2. Fundamental Principles  Under conditions where Coomassie Brilliant Blue G-250 is present in excess and at a constant concentration, varying protein concentrations in the solution result in different amounts of dye molecules transitioning from the 465 nm absorption form to the 595 nm form.  This transition follows a quantitative relationship.  Generally, as protein concentration increases, the absorbance of the reagent at 595 nm increases nearly linearly, allowing the Coomassie Brilliant Blue G-250 staining method to be used for protein quantitation

2.FundamentalPrinciplesCompared to the Lowry method, the advantages of theCoomassie Brilliant Blue method include: Simplicity:Requires only a singlereagentfor colordevelopment.Rapid reaction: Completes in a single step within 5minReducedinterference:Unaffected bymany compounds(e.g., sugars, buffers, reducing agents, and chelators) thatinterfere with the Lowrymethod. Limitations exist, such as imperfect linearity

Compared to the Lowry method, the advantages of the Coomassie Brilliant Blue method include:  Simplicity: Requires only a single reagent for color development.  Rapid reaction: Completes in a single step within 5 min.  Reduced interference: Unaffected by many compounds (e.g., sugars, buffers, reducing agents, and chelators) that interfere with the Lowry method.  Limitations exist, such as imperfect linearity. 2. Fundamental Principles

3.ExperimentalProcedures3.1PreparationofStandardCurvePrepare 6 clean, dry tubes and add reagents according to the following tableVolume ofVolume ofTotalCoomassieStandardProteinHO(mL)BrilliantBlueTube No.ProteinContent inAbs595Reagent(mL)Solution(mL)Tube (μg)511.0000.00025?0.2500.835?0.60.41005?40.40.6150055?0.20.82005?60.01.02500

3. Experimental Procedures 3.1 Preparation of Standard Curve Prepare 6 clean, dry tubes and add reagents according to the following table: Tube No. Volume of H₂O (mL) Volume of Standard Protein Solution (mL) Total Protein Content in Tube (μg) Coomassie Brilliant Blue Reagent (mL) Abs₅₉₅ 1 1.0 0 0 5 0.000 2 0.8 0.2 50 5 ? 3 0.6 0.4 100 5 ? 4 0.4 0.6 1500 5 ? 5 0.2 0.8 200 5 ? 6 0.0 1.0 2500 5 ?

3.Procedures> After adding all reagents, mix thoroughlyThe absorbance at 595 nm(Abss9s)canbe measured immediatelyusing a spectrophotometer.> Plot the standard curve with protein concentration as thex-axisandAbss9s asthey-axis

3. Procedures  After adding all reagents, mix thoroughly.  The absorbance at 595 nm (Abs₅₉₅) can be measured immediately using a spectrophotometer.  Plot the standard curve with protein concentration as the x-axis and Abs₅₉₅ as the y-axis

3.Experimental Procedures3.2Sample Measurement While preparing the standard curve, take another test tube, add1 mL of the sample to be tested, then add 5 mL of CoomassieBrilliant Blue reagent.>MixthoroughlyandmeasureAbss95>UsetheAbss9svalueto determinetheprotein concentrationofthe sample from the standard curve. To improve accuracy, perform a replicate measurement of thesample simultaneously

3.2 Sample Measurement  While preparing the standard curve, take another test tube, add 1 mL of the sample to be tested, then add 5 mL of Coomassie Brilliant Blue reagent.  Mix thoroughly and measure Abs₅₉₅.  Use the Abs₅₉₅ value to determine the protein concentration of the sample from the standard curve.  To improve accuracy, perform a replicate measurement of the sample simultaneously. 3. Experimental Procedures

Experiment2:the Identification of Reducing and TotalSugars in Sweet Potato Flour

Experiment 2: the Identification of Reducing and Total Sugars in Sweet Potato Flour

1.Objectives1.1 To identify reducing sugars and total sugars in sweet potato flour.1.2 To understand the fundamental principles of reducing and totalsugar determination.1.3 To learn the operation methods of spectrophotometric(colorimetry) determination of reducing sugars.1.4 To master the use of the spectrophotometer

1. Objectives 1.1 To identify reducing sugars and total sugars in sweet potato flour. 1.2 To understand the fundamental principles of reducing and total sugar determination. 1.3 To learn the operation methods of spectrophotometric (colorimetry) determination of reducing sugars. 1.4 To master the use of the spectrophotometer