Measuring Protein Concentration through Absorption Spectrophotometry

Size: px
Start display at page:

Download "Measuring Protein Concentration through Absorption Spectrophotometry"

Transcription

1 Measuring Protein Concentration through Absorption Spectrophotometry In this lab exercise you will learn how to homogenize a tissue to extract the protein, and then how to use a protein assay reagent to determine the concentration of protein in the sample. These are the initial steps in preparing your liver samples for gel electrophoresis and immunoblotting analysis of β-actin. Absorption spectrophotometry is a means for determining the concentration of a substance in solution. A dissolved substance will absorb light of specific wavelengths characteristic of that substance. When light of those wavelengths is passed through the sample, some of the light is absorbed by the solute, decreasing the amount of light that passes through the sample (Figure 1). A spectrophotometer is used to measure the difference in the amount of light entering and leaving the sample. The light that passes through the sample (not absorbed) is called transmitted light. This difference in the original and transmitted light is called the absorbance. Figure 1. Absorption of light as it passes through a solution. Absorption spectrophotometry can be used to study the properties of many types of biological molecules, such as pigments, enzymes, DNA, and many small organic molecules. This methodology can also be used to measure biological activities of living cells, such as enzyme activities and rates of photosynthesis. You will be using it to measure the concentration of protein in the liver homogenate that you prepare. What are some potential sources of error in absorbance measurements? Spectrophotometric measurements are affected by many factors, such as the type of solvent used, temperature, wavelength of light at which the measurements are made, and presence of impurities in the sample being studied. To eliminate absorbance due to the solvent, the spectrophotometer is always zeroed (tared) against the solvent. Another potential problem is light scattering is due to suspended particles. As shown below in Figure 2, the particulates will deflect light rays and cause an artificial increase in absorbance. To avoid light scattering, it is common practice to centrifuge the sample before measuring absorbance to remove particulates. Figure 2. Light scattering by suspended particles will lead to erroneous absorbance measurements. Protein Measurement and Absorption Spectrophotometry Page 1

2 How is absorption spectrophotometry used to measure protein concentration? Protein concentration is typically measured by combining a small sample of the homogenate with a chemical reagent that changes color in proportion to the amount of protein present. Several commercially prepared protein assay reagents are available, and for this lab we will use the BCA reagent. In the presence of protein turns a purple color the more protein present the deeper the color. The purplecolored product absorbs light best at 562 nm, the wavelength at which the measurements are made. Thus, 562 nm is the 8max (wavelength of maximum absorbance) for the BCA reagent in the presence of protein? The protein content of the liver homogenate will be determined by comparing the absorbance of homogenate sample to the absorption coefficient determined from a standard curve. A Standard Curve is prepared by plotting the absorbance of samples containing known concentrations of the standard protein having reacted with the (Figure 3). It doesn t matter which type of protein is used to create the standard curve because the BCA reagent reacts the same with all protein, so we will use a protein called bovine serum albumin (BSA). The slope (m) of the line (y = mx + b) estimates the absorbance coefficient for the BCA reagent after reacting with the protein. The slope can then be used to calculate the concentration of an unknown from it s absorbance as: c = Abs ) m. Figure 3. A BCA reagent standard curve is prepared by measuring the absorbance of samples with known amounts of BSA. What is an absorbance spectrum? How do we know that the BCA reagent has an 8max of 562nM? This can be determined by measuring the absorbance of a substance over a range of wavelengths to create an absorbance spectrum a plot of absorbance vs wavelength. (Figure 4). Since different molecules have unique absorbance properties, absorbance spectrum can also be used to identify an unknown substance and determine its structural characteristics. Figure 4 shows absorbance of the Bradford reagent, which also can be used to measure protein Figure 4. Absorbance spectra of Bradford concentration. Notice that the absorbance reagent in absence and presence of protein. spectrum of the reagent changes when it is bound to protein, and that the 8max shifts from 465nm to 595nm. Thus, 595nm is the best wavelength to make measurements since it will yield the highest absorbance values and thus the highest sensitivity; high sensitivity means that lower concentrations of protein can be detected and measured accurately. Protein Measurement and Absorption Spectrophotometry Page 2

3 Objectives The objectives of this lab exercise are for you to learn: $ how to prepare a tissue protein extract and use a protein assay reagent to measure the protein concentration. $ theory and practice of absorbance spectrophotometry. $ how to prepare and use a standard curve and absorption spectrum. Using the Genesys 20 Spectrophotometer 1. Turn on the spectrophotometer. Allow to warm up about 15 minutes (stabilizes light source). 2. Set to proper wavelength. 3. Transfer the blank (control) sample to a cuvette, place it in the sample holder, and set the absorbance to Replace the sample with the test sample and record the absorbance. Supplies 13 mm test tubes P-20 & P-200 pipeters ice bucket 2 ml & 5 ml pipets 3 cuvettes for homogenization: homogenization buffer 15 ml centrifuge tube sample of liver cryogenic storage vial Potter-Elvejhem homogenizer for protein assay 25 ml BCA protein reagent BSA protein standards 45 O C heater heating 2 spectrophotometer cuvettes Protein Measurement and Absorption Spectrophotometry Page 3

4 Procedures All samples, solutions, and utensils must be kept chilled in ice at all times, unless indicated otherwise. 1. Preparing the liver homogenate Homogenize the liver sample 1. Thaw a 0.5 g liver sample and transfer it to a Potter-Elvejhem homogenizer. 2. Add 5 ml of chilled homogenization buffer. 3. Homogenize with 3-4 passes of the pestle. Centrifuge the homogenate 4. Transfer your homogenate to a 15 ml high-speed centrifuge tube. Counterbalance against a tube of another group. Centrifuge at 12,000 rpm for 15 minutes in the SU34 rotor. 5. After the centrifugation, carefully transfer the supernatant to a labeled cryostorage vial held in an ice bucket. 2. Measuring the protein concentration of the homogenate *** The BCA assay must be performed for all of the samples at the same time*** *** The BCA reagent and assay tubes should not be chilled use at room temperature*** Dilute a portion of your homogenate. So that the protein concentration falls within the detection range of the BSA reagent, you must dilute a portion of your homogenate 50x with homogenization buffer to a final volume of 5 ml (5000 :l). Record these values in your lab notebook: Volume of homogenate needed = 5000 :l ) 50 = :l Volume of buffer needed = 5000 :l (above value) = :l 1. Measuring the buffer with a 5 ml pipet and the homogenate with a P-200 pipetter, combine and mix the diluted sample in a 13 mm test tube. Keep this diluted sample on ice. Label the standard curve and homogenate assay tubes. 2. Label the 6 tubes to receive the BSA standard protein 0", 0.2", 0.4", 0.6", 0.8", and 1.0" :g/:l. 3. Label the 4 tubes to receive sample homogenate Blank, #1", #2", #3". Add the BSA standard curve assay samples to their tubes. The BSA protein is provided in vials containing BSA in concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 :g/:l. 4. Add 100:L of each solution (using a P-200 pipetter) to separate test tubes. Accuracy in pipetting is crucial, so be sure to place the sample in the bottom of the tube. Protein Measurement and Absorption Spectrophotometry Page 4

5 Add the three replica tissue homogenate samples to their tubes. 5. Add 100 :L of the 50X diluted sample to each test tube labeled #1, #2, and #3. 6. Add 100 :L of homogenization buffer to the tube labeled blank. Add 2 ml of the BCA reagent to all of the tubes. 7. Using a 2 ml pipet, carefully but expediently add the BCA reagent to each tube. Heat the samples 45 o C for 15 minutes. 8. Place the tubes in the heat source provided (might be an water bath or heater block). 9. Remove the tubes and allow to cool for 5 10 minutes. Take the absorbance measurements for the standard curve. This can be done using only 1 cuvette. Transfer samples to and from the cuvette using Pasteur Pipets 10. Using the sample containing 0 :g/:l BSA, zero the spectrophotometer. Then transfer the sample back to its original tube. 11. Using the same cuvette, record the absorbance measurements progressing upward from the 0.2 to the 1.0 :g/:l sample. Using before measuring the absorbance of the BSA and liver samples. The 0 :g/:l BSA sample will serve as the blank for the standard curve samples, and the tube containing only homogenization buffer will serve as the blank for the liver samples. Save the 0 and 0.4 :g/ml samples from the standard curve for Part 3 Take the absorbance measurements for the three homogenate replicas. 12. Using the same basic procedure as described above, but a new cuvette, measure the absorbance of the liver homogenate samples. Store your liver homogenate 13. Label the cryogenic storage vial with your name, date and tissue. 14. Chill the vial in your ice bucket, and then transfer your homogenate (not the 50X diluted sample) to the vial with a Pasteur pipet. 15. Place your vial in the designated container in the -80EC freezer. Protein Measurement and Absorption Spectrophotometry Page 5

6 3. Determining the Absorbance Spectrum of the BCA Reagent in the Presence and Absence of Protein. The objective of this procedure is to determine the absorbance spectrum of BCA reagent in the presence and absence of protein, determine the wavelength of light at which maximum absorbance occurs (8 max ), and compare this to the 8 max given by the manufacturer. Supplies 3 cuvettes 0 :g/:l BSA and 0.4 :g/:l BSA samples from standard curve Procedure The absorbance spectrum of the BCA reagent will be determined by measuring the absorbance of the sample at many different wavelengths, zeroing the spectrophotometer against the blank each time. 1. Using 3 cuvettes, place dh 2 O into one, the 0 :g/:l BSA sample into another, and the 0.4 :g/:l BSA sample into third cuvette. 2. Take absorbance measurements of the two BCA samples at 20 nm intervals starting at a wavelength of 420 nm and ending at 700 nm. Each reading must be made after first zeroing the machine against the dh 2 O blank. Record your absorbance measurements in Table 3. Note: there will be an initial high absorbance at short wavelengths; this is not the peak that you are will be looking for. 3. To narrow the wavelength at which the maximum absorbance (8 max ) occurs for the BCA + protein sample, take measurements at 5 nm intervals in the region of the 8 max. Record your absorbance measurements in Plot the full absorbance spectra ( nm) for the BCA samples with and without protein on a single graph using Excel. Plot the data for the 5 nm increments on a separate graph, and mark the 8 max. Protein Measurement and Absorption Spectrophotometry Page 6

7 Calculations and Graphing 1. Determine the absorption coefficient for the BCA reagent used with BSA. Graph the standard curve (including 0 :g) using Excel. Check the set intercept = 0" under Trendline Options this assures that the trendline will pass through 0,0 The Absorbance coefficient = slope of trendline 2. Calculate the protein concentration in the homogenates. Use the Beer-Lambert law to determine the protein concentration for the three liver replicas and then average the values. Remembering that the homogenate was diluted 50X prior to performing the BCA assay, the concentration of original homogenate (:g/:l) can now be calculated: Protein concentration in the homogenate = Avg. :g protein x dilution factor 3. Calculations for the Electrophoresis lab For the upcoming electrophoresis lab exercise, you will need to work with a volume of your homogenate that contains 50 :g of protein; calculate this volume now: volume of homogenate = 50 :g ) Protein concentration in the homogenate 4. Graph the absorbance spectrum data as two separate graphs, not as a combined data set. Protein Measurement and Absorption Spectrophotometry Page 7

8 Protein Measurement and Absorption Spectrophotometry Page 8

Studying Photosynthetic Electron Transport through the Hill Reaction

Studying Photosynthetic Electron Transport through the Hill Reaction Studying Photosynthetic Electron Transport through the Hill Reaction In this lab exercise you will learn techniques for studying electron transport. While our research system will be chloroplasts and photosynthetic

More information

Using the Spectrophotometer

Using the Spectrophotometer Using the Spectrophotometer Introduction In this exercise, you will learn the basic principals of spectrophotometry and and serial dilution and their practical application. You will need these skills to

More information

EXPERIMENT 11 UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of Potassium Permanganate Solutions.

EXPERIMENT 11 UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of Potassium Permanganate Solutions. EXPERIMENT 11 UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of Potassium Permanganate Solutions. Outcomes After completing this experiment, the student should be able to: 1. Prepare

More information

Biology 309 Lab Notebook

Biology 309 Lab Notebook Name: Biology 309 Lab Notebook This is a guided lab notebook for you to keep well-organized notes about procedures and record experimental data for experiments as they are performed. It is guided because,

More information

Chem 131A: Absorbance of Riboflavin

Chem 131A: Absorbance of Riboflavin Chem 131A: Absorbance of Riboflavin Purpose: The purpose of this experiment is to: 1) Familiarize the student with the use of the HP 8452 diode array spectrophotometer, 2) examine the limitations of the

More information

Application Note: Absorbance

Application Note: Absorbance Units Units Theory of absorbance Light absorption occurs when atoms or molecules take up the energy of a photon of light, thereby reducing the transmission of light as it is passed through a sample. Light

More information

2 Spectrophotometry and the Analysis of Riboflavin

2 Spectrophotometry and the Analysis of Riboflavin 2 Spectrophotometry and the Analysis of Riboflavin Objectives: A) To become familiar with operating the Platereader; B) to learn how to use the Platereader in determining the absorption spectrum of a compound

More information

Lab 2. Spectrophotometric Measurement of Glucose

Lab 2. Spectrophotometric Measurement of Glucose Lab 2 Spectrophotometric Measurement of Glucose Objectives 1. Learn how to use a spectrophotometer. 2. Produce a glucose standard curve. 3. Perform a glucose assay. Safety Precautions Glucose Color Reagent

More information

RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit

RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit RayBio Creatine Kinase (CK) Activity Colorimetric Assay Kit User Manual Version 1.0 May 28, 2014 RayBio Creatine Kinase Activity Colorimetric Assay (Cat#: 68CL-CK-S100) RayBiotech, Inc. We Provide You

More information

Creatine Kinase Activity Assay Kit (Colorimetric)

Creatine Kinase Activity Assay Kit (Colorimetric) ab155901 Creatine Kinase Activity Assay Kit (Colorimetric) Instructions for Use For the sensitive and accurate measurement of Creatine Kinase activity in various samples. This product is for research use

More information

Chemistry 111 Lab: Intro to Spectrophotometry Page E-1

Chemistry 111 Lab: Intro to Spectrophotometry Page E-1 Chemistry 111 Lab: Intro to Spectrophotometry Page E-1 SPECTROPHOTOMETRY Absorption Measurements & their Application to Quantitative Analysis study of the interaction of light (or other electromagnetic

More information

ENZYME KINETICS ENZYME-SUBSTRATE PRODUCTS

ENZYME KINETICS ENZYME-SUBSTRATE PRODUCTS ENZYME KINETICS INTRODUCTION The study of reaction rates catalyzed by enzymes and the factors affecting them is generally referred to as enzyme kinetics. The basic components of an enzyme catalyzed reaction

More information

Factors Affecting Enzyme Activity

Factors Affecting Enzyme Activity INTRODUCTION Factors Affecting Enzyme Activity The chemical reactions occurring in living things are controlled by enzymes. An enzyme is a protein in the cell which lowers the activation energy of a catalyzed

More information

Austin Peay State University Department of Chemistry Chem 1111. The Use of the Spectrophotometer and Beer's Law

Austin Peay State University Department of Chemistry Chem 1111. The Use of the Spectrophotometer and Beer's Law Purpose To become familiar with using a spectrophotometer and gain an understanding of Beer s law and it s relationship to solution concentration. Introduction Scientists use many methods to determine

More information

Colorimetry Extinction coefficient (ε) Lambda max (λ max ) Qualitative vs. quantitative analysis

Colorimetry Extinction coefficient (ε) Lambda max (λ max ) Qualitative vs. quantitative analysis Lab Week 2 - Spectrophotometry Purpose: Introduce students to the use of spectrophotometry for qualitative (what is it) and quantitative (how much is there of it) analysis of biological samples and molecules.

More information

6 Characterization of Casein and Bovine Serum Albumin

6 Characterization of Casein and Bovine Serum Albumin 6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function

More information

Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution.

Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Introduction: The determination of protein concentration is frequently required in biochemical work. Several methods

More information

Catalase. ***You will be working with hot water, acids and bases in this laboratory*** ****Use Extreme Caution!!!****

Catalase. ***You will be working with hot water, acids and bases in this laboratory*** ****Use Extreme Caution!!!**** AP BIOLOGY BIOCHEMISTRY ACTIVITY #9 NAME DATE HOUR CATALASE LAB INTRODUCTION Hydrogen peroxide (H 2 O 2 ) is a poisonous byproduct of metabolism that can damage cells if it is not removed. Catalase is

More information

Laboratory 5: Properties of Enzymes

Laboratory 5: Properties of Enzymes Laboratory 5: Properties of Enzymes Technical Objectives 1. Accurately measure and transfer solutions with pipettes 2. Use a Spectrophotometer to study enzyme action. 3. Properly graph a set of data. Knowledge

More information

A Beer s Law Experiment

A Beer s Law Experiment A Beer s Law Experiment Introduction There are many ways to determine concentrations of a substance in solution. So far, the only experiences you may have are acid-base titrations or possibly determining

More information

Upon completion of this lab, the student will be able to:

Upon completion of this lab, the student will be able to: 1 Learning Outcomes EXPERIMENT B4: CHEMICAL EQUILIBRIUM Upon completion of this lab, the student will be able to: 1) Analyze the absorbance spectrum of a sample. 2) Calculate the equilibrium constant for

More information

Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C

Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C I. Introduction: Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N- phosphotransferase

More information

Spectrophotometry and the Beer-Lambert Law: An Important Analytical Technique in Chemistry

Spectrophotometry and the Beer-Lambert Law: An Important Analytical Technique in Chemistry Spectrophotometry and the Beer-Lambert Law: An Important Analytical Technique in Chemistry Jon H. Hardesty, PhD and Bassam Attili, PhD Collin College Department of Chemistry Introduction: In the last lab

More information

QuickZyme Soluble Collagen Assay

QuickZyme Soluble Collagen Assay QuickZyme Soluble Collagen Assay Version June 2012 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES This package insert must be read in its entirety before using this product. Introduction Collagen

More information

Lab #11: Determination of a Chemical Equilibrium Constant

Lab #11: Determination of a Chemical Equilibrium Constant Lab #11: Determination of a Chemical Equilibrium Constant Objectives: 1. Determine the equilibrium constant of the formation of the thiocyanatoiron (III) ions. 2. Understand the application of using a

More information

Enzymes: Amylase Activity in Starch-degrading Soil Isolates

Enzymes: Amylase Activity in Starch-degrading Soil Isolates Enzymes: Amylase Activity in Starch-degrading Soil Isolates Introduction This week you will continue our theme of industrial microbiologist by characterizing the enzyme activity we selected for (starch

More information

Reaction Stoichiometry and the Formation of a Metal Ion Complex

Reaction Stoichiometry and the Formation of a Metal Ion Complex Reaction Stoichiometry and the Formation of a Metal Ion Complex Objectives The objectives of this laboratory are as follows: To use the method of continuous variation to determine the reaction stoichiometry

More information

Experiment 7 (Lab Period 8) Quantitative Determination of Phosphatase Activity

Experiment 7 (Lab Period 8) Quantitative Determination of Phosphatase Activity Experiment 7 (Lab Period 8) Quantitative Determination of Phosphatase Activity Phosphatases are enzymes that catalyze the hydrolysis of organic-phosphate compounds, releasing inorganic phosphate from the

More information

Determining the Quantity of Iron in a Vitamin Tablet. Evaluation copy

Determining the Quantity of Iron in a Vitamin Tablet. Evaluation copy Determining the Quantity of Iron in a Vitamin Tablet Computer 34 As biochemical research becomes more sophisticated, we are learning more about the role of metallic elements in the human body. For example,

More information

Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40

Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40 Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40 Note: there is a second document that goes with this one! 2046 - Absorbance Spectrophotometry - Calibration Curve Procedure. The second document

More information

Lab 2 Biochemistry. Learning Objectives. Introduction. Lipid Structure and Role in Food. The lab has the following learning objectives.

Lab 2 Biochemistry. Learning Objectives. Introduction. Lipid Structure and Role in Food. The lab has the following learning objectives. 1 Lab 2 Biochemistry Learning Objectives The lab has the following learning objectives. Investigate the role of double bonding in fatty acids, through models. Developing a calibration curve for a Benedict

More information

Spectrophotometry Practical Lesson on Medical Chemistry and Biochemistry

Spectrophotometry Practical Lesson on Medical Chemistry and Biochemistry Spectrophotometry Practical Lesson on Medical Chemistry and Biochemistry General Medicine Jiřina Crkovská (translated by Jan Pláteník) 2010/2011 1 Spectrophotometry is one of the most widely used instrumental

More information

The Determination of an Equilibrium Constant

The Determination of an Equilibrium Constant The Determination of an Equilibrium Constant Chemical reactions occur to reach a state of equilibrium. The equilibrium state can be characterized by quantitatively defining its equilibrium constant, K

More information

Experiment 13H THE REACTION OF RED FOOD COLOR WITH BLEACH 1

Experiment 13H THE REACTION OF RED FOOD COLOR WITH BLEACH 1 Experiment 13H FV 1/25/2011(2-run) THE REACTION OF RED FOOD COLOR WITH BLEACH 1 PROBLEM: Determine the rate law for the chemical reaction between FD&C Red Dye #3 and sodium hypochlorite. LEARNING OBJECTIVES:

More information

THE ACTIVITY OF LACTASE

THE ACTIVITY OF LACTASE THE ACTIVITY OF LACTASE Lab VIS-8 From Juniata College Science in Motion Enzymes are protein molecules which act to catalyze the chemical reactions in living things. These chemical reactions make up the

More information

Measuring Manganese Concentration Using Spectrophotometry

Measuring Manganese Concentration Using Spectrophotometry Measuring Manganese Concentration Using Spectrophotometry Objectives To use spectroscopy to determine the amount of Manganese is an unknown sample. Scenario Your have just joined a "Green Team" at SMC

More information

Activity Sheets Enzymes and Their Functions

Activity Sheets Enzymes and Their Functions Name: Date: Activity Sheets Enzymes and Their Functions amylase What are Enzymes? starch glucose Enzymes are compounds that assist chemical reactions by increasing the rate at which they occur. For example,

More information

Experiment 2 Kinetics II Concentration-Time Relationships and Activation Energy

Experiment 2 Kinetics II Concentration-Time Relationships and Activation Energy 2-1 Experiment 2 Kinetics II Concentration-Time Relationships and Activation Energy Introduction: The kinetics of a decomposition reaction involving hydroxide ion and crystal violet, an organic dye used

More information

Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105

Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105 Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105 Background Gold nanoparticle conjugates have been widely used in biological research and biosensing applications.

More information

LAB TOPIC 4: ENZYMES. Enzyme catalyzed reactions can be expressed in the following way:

LAB TOPIC 4: ENZYMES. Enzyme catalyzed reactions can be expressed in the following way: LAB TOPIC 4: ENZYMES Objectives Define enzyme and describe the activity of enzymes in cells. Discuss the effects of varying enzyme concentrations on the rate of enzyme activity. Discuss the effects of

More information

TOTAL PROTEIN FIBRINOGEN

TOTAL PROTEIN FIBRINOGEN UNIT: Proteins 16tproteins.wpd Task Determination of Total Protein, Albumin and Globulins Objectives Upon completion of this exercise, the student will be able to: 1. Explain the ratio of albumin and globulin

More information

The Determination of an Equilibrium Constant

The Determination of an Equilibrium Constant The Determination of an Equilibrium Constant Computer 10 Chemical reactions occur to reach a state of equilibrium. The equilibrium state can be characterized by quantitatively defining its equilibrium

More information

Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit

Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit Catalog No. CSB-E15852c (96T) This immunoassay kit allows for the in vitro quantitative determination of canine CK-MB concentrations in serum and plasma.

More information

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 2:CHLOROPLASTS AND PHOTOREDUCTION In this laboratory we will purify chloroplasts from spinach by differential centrifugation, then

More information

Table of Content. Enzymes and Their Functions Teacher Version 1

Table of Content. Enzymes and Their Functions Teacher Version 1 Enzymes and Their Functions Jeisa Pelet, Cornell University Carolyn Wilczynski, Binghamton High School Cornell Learning Initiative in Medicine and Bioengineering (CLIMB) Table of Content Title Page Abstract..

More information

ab83369 Alkaline Phosphatase Assay kit (Colorimetric)

ab83369 Alkaline Phosphatase Assay kit (Colorimetric) ab83369 Alkaline Phosphatase Assay kit (Colorimetric) Instructions for use: For the rapid, sensitive and accurate measurement of Alkaline Phosphatase in various samples. This product is for research use

More information

Benchtop Mitochondria Isolation Protocol

Benchtop Mitochondria Isolation Protocol Benchtop Mitochondria Isolation Protocol Note: Specific protocols are available for the following products: MS850 Mitochondria Isolation Kit for Rodent Tissue MS851 Mitochondria Isolation Kit for Rodent

More information

Creatine Kinase Microplate Assay Kit User Manual

Creatine Kinase Microplate Assay Kit User Manual Creatine Kinase Microplate Assay Kit User Manual Catalog # CAK1045 Detection and Quantification of Creatine Kinase (CK) Activity in Urine, Serum, Plasma, Tissue extracts, Cell lysate, Cell culture media

More information

Human Free Testosterone(F-TESTO) ELISA Kit

Human Free Testosterone(F-TESTO) ELISA Kit Human Free Testosterone(F-TESTO) ELISA Kit Catalog Number. MBS700040 For the quantitative determination of human free testosterone(f-testo) concentrations in serum, plasma. This package insert must be

More information

EXPERIMENT 5. Molecular Absorption Spectroscopy: Determination of Iron With 1,10-Phenanthroline

EXPERIMENT 5. Molecular Absorption Spectroscopy: Determination of Iron With 1,10-Phenanthroline EXPERIMENT 5 Molecular Absorption Spectroscopy: Determination of Iron With 1,10-Phenanthroline UNKNOWN Submit a clean, labeled 100-mL volumetric flask to the instructor so that your unknown iron solution

More information

10/5/06 Lab 3. Protein Determination. Lab 3. PROTEIN DETERMINATION

10/5/06 Lab 3. Protein Determination. Lab 3. PROTEIN DETERMINATION 10/5/06 Lab 3. Protein Determination Lab 3. PROTEIN DETERMINATION I. INTRODUCTION Reading in Biology, 6 th ed. By Campbell: PROTEINS-MANY STRUCTURES, MANY FUNCTIONS p. 71-80 Blood is a connective tissue

More information

Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA

Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA KAMIYA BIOMEDICAL COMPANY Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA For the quantitative determination of mouse CKMB in serum, plasma, cell culture fluid and other biological fluids Cat. No. KT-57681

More information

CHEM 161: Beer s Law and Analysis of a Sports Drink

CHEM 161: Beer s Law and Analysis of a Sports Drink CHEM 161: Beer s Law and Analysis of a Sports Drink Introduction Although sunlight appears white, it contains a spectrum of colors. A rainbow actually shows this range of colors in visible light: violet,

More information

HiPer Ion Exchange Chromatography Teaching Kit

HiPer Ion Exchange Chromatography Teaching Kit HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for

More information

What s in the Mix? Liquid Color Spectroscopy Lab (Randy Landsberg & Bill Fisher)

What s in the Mix? Liquid Color Spectroscopy Lab (Randy Landsberg & Bill Fisher) What s in the Mix? Liquid Color Spectroscopy Lab (Randy Landsberg & Bill Fisher) Introduction: There is more to a color than a name. Color can tell us lots of information. In this lab you will use a spectrophotometer

More information

HiPer Total RNA Extraction Teaching Kit

HiPer Total RNA Extraction Teaching Kit HiPer Total RNA Extraction Teaching Kit Product Code: HTBM012 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1 hour Agarose Gel Electrophoresis: 1 hour Storage Instructions:

More information

MTT Cell Proliferation Assay

MTT Cell Proliferation Assay ATCC 30-1010K Store at 4 C This product is intended for laboratory research purposes only. It is not intended for use in humans, animals or for diagnostics. Introduction Measurement of cell viability and

More information

EFFECT OF SALT ON CELL MEMBRANES

EFFECT OF SALT ON CELL MEMBRANES EFFECT OF SALT ON CELL MEMBRANES LAB CELL 2 INTRODUCTION A eukaryotic cell, a cell with a nucleus, not only has a plasma membrane as its external boundary, but it also has a variety of membranes that divide

More information

Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit

Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Catalog No: E0479r 96 Tests Operating instructions www.eiaab.com FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH

More information

Plant Genomic DNA Extraction using CTAB

Plant Genomic DNA Extraction using CTAB Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however

More information

ATOMIC ABSORTION SPECTROSCOPY: rev. 4/2011 ANALYSIS OF COPPER IN FOOD AND VITAMINS

ATOMIC ABSORTION SPECTROSCOPY: rev. 4/2011 ANALYSIS OF COPPER IN FOOD AND VITAMINS 1 ATOMIC ABSORTION SPECTROSCOPY: rev. 4/2011 ANALYSIS OF COPPER IN FOOD AND VITAMINS Buck Scientific Atomic Absorption Spectrophotometer, Model 200 Atomic absorption spectroscopy (AAS) has for many years

More information

PROTEINS (LOWRY) PROTOCOL

PROTEINS (LOWRY) PROTOCOL 1 PROTEINS (LOWRY) PROTOCOL 1. INTRODUCTION The Lowry Assay: Protein by Folin Reaction (Lowry et al., 1951) has been the most widely used method to estimate the amount of proteins (already in solution

More information

2C: One in a Million. Part 1: Making solutions. Name: Section: Date: Materials

2C: One in a Million. Part 1: Making solutions. Name: Section: Date: Materials Name: Section: Date: 2C: One in a Million Drinking water can contain up to 1.3 parts per million (ppm) of copper and still be considered safe. What does parts per million mean? Both living things and the

More information

SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual

SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Toll Free: 866-252-7771 752A Lincoln Blvd. Phone: 732-469-7771 Fax: 732-469-7782 Middlesex, NJ 08846 Web: www.purebiotechllc.com SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Product: SOLIDscript

More information

HBV Quantitative Real Time PCR Kit

HBV Quantitative Real Time PCR Kit Revision No.: ZJ0002 Issue Date: Aug 7 th, 2008 HBV Quantitative Real Time PCR Kit Cat. No.: HD-0002-01 For Use with LightCycler 1.0/LightCycler2.0/LightCycler480 (Roche) Real Time PCR Systems (Pls ignore

More information

For the rapid, sensitive and accurate measurement of ATP in various samples.

For the rapid, sensitive and accurate measurement of ATP in various samples. ab83355 ATP Assay Kit (Colorimetric/ Fluorometric) Instructions for use: For the rapid, sensitive and accurate measurement of ATP in various samples. This product is for research use only and is not intended

More information

Hydrogen Peroxide Cell-Based Assay Kit

Hydrogen Peroxide Cell-Based Assay Kit Hydrogen Peroxide Cell-Based Assay Kit Item No. 600050 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION

More information

Protein expression in the life cycle of bean beetles (Callosobruchus maculatus)

Protein expression in the life cycle of bean beetles (Callosobruchus maculatus) Protein expression in the life cycle of bean beetles (Callosobruchus maculatus) Pre laboratory Preparation Instructor s Notes You will need a number of cultures of bean beetles at various life stages.

More information

Using Spectrophotometers to Examine Photosynthetic Rates Under Various Qualities of Light

Using Spectrophotometers to Examine Photosynthetic Rates Under Various Qualities of Light Purdue GK-12 Lesson Plan 2006-07 Using Spectrophotometers to Examine Photosynthetic Rates Under Various Qualities of Light Purdue University GK-12 2006-2007 Lead developer and contact: Amanda Deering Purdue

More information

BY UV SPECTROPHOTOMETRY

BY UV SPECTROPHOTOMETRY 1 of 9 1. PURPOSE This Standard Operating Procedure describes the method for measuring the concentration of viral particles using UV/Vis spectrophotometry at 260 and 280 nm. 2. SCOPE This procedure is

More information

Spectrophotometer - Milton Roy Spectronic 21D or equivalent.

Spectrophotometer - Milton Roy Spectronic 21D or equivalent. 3M COMPANY OCCUPATIONAL HEALTH AND ENVIRONMENTAL SAFETY DIVISION DETERMINATION OF FORMALDEHYDE VAPORS IN AIR USING 3M 3721 FORMALDEHYDE MONITORS May, 2002 SCOPE This procedure covers the method of collecting

More information

Western Blot Protocol Protein isolation

Western Blot Protocol Protein isolation Western Blot Protocol Protein isolation A. Preparation of cell lysates. - Preparation of materials: -Dial the microcentrifuge temperature control setting to 4 C -Prepare a bucket of ice -Prepare lysis

More information

DNA Electrophoresis Lesson Plan

DNA Electrophoresis Lesson Plan DNA Electrophoresis Lesson Plan Primary Learning Outcomes: Students will learn how to properly load a well in an agarose gel. Students will learn how to analyze the results of DNA electrophoresis. Students

More information

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Catalog Number. CSB-E14405r For the quantitative determination of rat creatine kinase MM isoenzyme (CK-MM) concentrations in serum, plasma and tissue

More information

Bovine Vitamin B12 (VB12) ELISA Kit

Bovine Vitamin B12 (VB12) ELISA Kit Bovine Vitamin B12 (VB12) ELISA Kit Catalog Number. CSB-E14089B For the quantitative determination of endogenic bovine vitamin B12 (VB12) concentrations in serum, plasma, tissue homogenates. This package

More information

Nucleic Acid Purity Assessment using A 260 /A 280 Ratios

Nucleic Acid Purity Assessment using A 260 /A 280 Ratios Nucleic Acid Purity Assessment using A 260 /A 280 Ratios A common practice in molecular biology is to perform a quick assessment of the purity of nucleic acid samples by determining the ratio of spectrophotometric

More information

Enzyme Action: Testing Catalase Activity

Enzyme Action: Testing Catalase Activity Enzyme Action: Testing Catalase Activity Experiment 6A Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities

More information

University of Wisconsin Chemistry 524 Spectroscopic Applications (GFAA, ICP, UV/Vis, Fluorescence)

University of Wisconsin Chemistry 524 Spectroscopic Applications (GFAA, ICP, UV/Vis, Fluorescence) University of Wisconsin Chemistry 524 Spectroscopic Applications (GFAA, ICP, UV/Vis, Fluorescence) For this laboratory exercise, you will explore a variety of spectroscopic methods used in an analytical

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

Reaction of Blue Food Dye with Bleach

Reaction of Blue Food Dye with Bleach Exercise 2 Reaction of Blue Food Dye with Bleach 2 Introduction In the experiment, you will study the rate of the reaction of FD&C Blue #1 (Blue #1 is denoted by E number E133 in food stuff) with sodium

More information

Evaluation copy. Case File 9. A Killer Cup of Coffee? GlobalTech manager dies

Evaluation copy. Case File 9. A Killer Cup of Coffee? GlobalTech manager dies Case File 9 Killer Cup of Coffee: Using colorimetry to determine concentration of a poison Determine the concentration of cyanide in the solution. A Killer Cup of Coffee? SOUTH PAINTER, Tuesday: It was

More information

INTERNATIONAL OLIVE COUNCIL

INTERNATIONAL OLIVE COUNCIL INTERNATIONAL OLIVE COUNCIL COI/T.20/Doc. No 19/Rev. 3 February 2015 ENGLISH Original: ENGLISH Príncipe de Vergara, 154 28002 Madrid España Telef.: +34 915 903 638 Fax: +34 915 631 263 - e-mail: iooc@internationaloliveoil.org

More information

ab185915 Protein Sumoylation Assay Ultra Kit

ab185915 Protein Sumoylation Assay Ultra Kit ab185915 Protein Sumoylation Assay Ultra Kit Instructions for Use For the measuring in vivo protein sumoylation in various samples This product is for research use only and is not intended for diagnostic

More information

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2 Uscn Life Science Inc. Wuhan Website: www.uscnk.com Phone: +86 27 84259552 Fax: +86 27 84259551 E-mail: uscnk@uscnk.com ELISA Kit for Human Prostaglandin E1(PG-E1) Instruction manual Cat. No.: E0904Hu

More information

Solubility Product Constants

Solubility Product Constants Solubility Product Constants PURPOSE To measure the solubility product constant (K sp ) of copper (II) iodate, Cu(IO 3 ) 2. GOALS 1 To measure the molar solubility of a sparingly soluble salt in water.

More information

IgM ELISA. For the quantitative determination of IgM in human serum and plasma. For Research Use Only. Not For Use In Diagnostic Procedures.

IgM ELISA. For the quantitative determination of IgM in human serum and plasma. For Research Use Only. Not For Use In Diagnostic Procedures. IgM ELISA For the quantitative determination of IgM in human serum and plasma For Research Use Only. Not For Use In Diagnostic Procedures. Please read carefully due to Critical Changes, e.g., Calibrator

More information

NNIN Nanotechnology Education

NNIN Nanotechnology Education NNIN Nanotechnology Education Student Guide Part 1: Silver Nanoparticle Synthesis and Spectroscopy Introduction: In this lab you will synthesize silver nanoparticles one of the most commonly used nanoparticles

More information

Glycolysis Cell-Based Assay Kit

Glycolysis Cell-Based Assay Kit Glycolysis Cell-Based Assay Kit Item No. 600450 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION

More information

Measuring Cell Viability/Cytotoxicity: Cell Counting Kit-F

Measuring Cell Viability/Cytotoxicity: Cell Counting Kit-F Introduction The Cell Counting Kit-F is a fluorometic assay for the determination of viable cell numbers. Calcein-AM in this kit passes through the cell membrane and is hydrolized by the esterase in the

More information

Determining the Free Chlorine Content of Swimming Pool Water. HOCl H + + OCl. Evaluation copy

Determining the Free Chlorine Content of Swimming Pool Water. HOCl H + + OCl. Evaluation copy Determining the Free Chlorine Content of Swimming Pool Water Computer 33 Physicians in the nineteenth century used chlorine water as a disinfectant. Upon the discovery that certain diseases were transmitted

More information

Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07

Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07 Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07 TABLE OF CONTENTS GENERAL INFORMATION... 2 Product Description... 2 Procedure Overview... 2 Kit Contents, Storage and Shelf Life... 3

More information

UNIT: Total and Direct Bilirubin

UNIT: Total and Direct Bilirubin UNIT: Total and Direct Bilirubin 13bili.wpd Task Determination of total and direct bilirubin. Objectives Upon completion of this exercise, the student will be able to: 1. Explain formation, excretion,

More information

Quantifying Bacterial Concentration using a Calibrated Growth Curve

Quantifying Bacterial Concentration using a Calibrated Growth Curve BTEC 4200 Lab 2. Quantifying Bacterial Concentration using a Calibrated Growth Curve Background and References Bacterial concentration can be measured by several methods, all of which you have studied

More information

Instructions and lab report for the practical lesson on biochemistry. Task 1: Qualitative estimation of protein in CSF (Pandy s test)

Instructions and lab report for the practical lesson on biochemistry. Task 1: Qualitative estimation of protein in CSF (Pandy s test) Date... Name... Group... Instructions and lab report for the practical lesson on biochemistry Topic: Biochemical examination of cerebrospinal fluid Task 1: Qualitative estimation of protein in CSF (Pandy

More information

ENZYME ACTION: TESTING CATALASE ACTIVITY

ENZYME ACTION: TESTING CATALASE ACTIVITY ENZYME ACTION: TESTING CATALASE ACTIVITY LAB ENZ 1.CALC From Biology with Calculators, Vernier Software & Technology, 2000 INTRODUCTION Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically.

More information

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit

Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Catalog Number. CSB-E14405r For the quantitative determination of rat creatine kinase MM isoenzyme (CK-MM) concentrations in serum, plasma, tissue homogenates.

More information

Concentrations and Dilutions of Food Dyes

Concentrations and Dilutions of Food Dyes Concentrations and Dilutions of Food Dyes Learning Goals: 1. Develop an understanding of the use of volumetric glassware. 2. Prepare a series of dye solutions of known concentrations. 3. Explore the relationship

More information

EFFECT OF ALCOHOL ON CELL MEMBRANES

EFFECT OF ALCOHOL ON CELL MEMBRANES EFFECT OF ALCOHOL ON CELL MEMBRANES LAB CELL 1 INTRODUCTION A eukaryotic cell, a cell with a nucleus, not only has a plasma membrane as its external boundary, but it also has a variety of membranes that

More information

Chapter 5 -- The Spectrophotometric Determination of the ph of a Buffer. NAME: Lab Section: Date: Sign-Off:

Chapter 5 -- The Spectrophotometric Determination of the ph of a Buffer. NAME: Lab Section: Date: Sign-Off: Chapter 5 -- The Spectrophotometric Determination of the ph of a Buffer NAME: Lab Section: Date: Sign-Off: Chapter 5 -- The Spectrophotometric Determination of the ph of a Buffer Introduction Weak acids,

More information

Chlorine, Total. DPD Method 1 Method 10101 0.09 to 5.00 mg/l Cl 2 Test 'N Tube Vials. Test preparation. Instrument-specific information

Chlorine, Total. DPD Method 1 Method 10101 0.09 to 5.00 mg/l Cl 2 Test 'N Tube Vials. Test preparation. Instrument-specific information Chlorine, Total DOC316.53.01028 DPD Method 1 Method 10101 0.09 to 5.00 mg/l Cl 2 Test 'N Tube Vials Scope and application: For testing higher levels of total (free plus combined) chlorine in drinking water,

More information