Absorption Spectrophotometry and Protein Measurement

Absorption spectrophotometry is one of the most powerful techniques available for investigating cells and cellular components. 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. Spectrophotometry can also be used to measure biological activities of living cells, such as enzyme activities and rates of photosynthesis. Absorption spectrophotometry measures the characteristic of substances to absorb electromagnetic radiation. Electromagnetic radiation occurs in a spectrum of wavelengths that extends from gamma rays of very short wavelengths to radio waves that have wavelengths measured in meters (Figure 1). The region of the electromagnetic spectrum that is most useful for the investigation of biological systems lies between wavelengths of about 240 nm and 800 nm. This range includes part of the ultraviolet region (from 190 to 380 nm) and the region of visible radiation, called "visible light" (from 380 to 750 nm). To put the wavelengths of light in proper perspective remember that: 1 nm = 1 nanometer = 1 x 10-3 micrometer (:m) = 1 x 10-6 millimeter (mm) = 1 x 10-9 meter

Figure 1. The electromagnetic spectrum.

Objectives
The objectives of this lab exercise are for you to learn: $ the theory and practice of using absorbance spectrophotometry. $ how to prepare a standard curve and absorption spectrum. $ how to use the Beer-Lambert equation. $ methodology of tissue homogenization. $ how to measure the protein concentration of a tissue homogenate.

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Transmittance vs Absorbance
Spectrophotometry is a means for determining the concentration of a substance in solution. A dissolved substance will absorb light of specific wavelengths and thus decrease the amount of light that passes through the sample. By using light of the appropriate wavelength, the concentration of an absorbing substance can be determined by comparing the intensity of the light before and after it passes through the solution. The light that passes through the sample (not absorbed) is called transmitted light (Figure 2).

Figure 2. Absorption of light as it passes through a solution. Percent transmittance (%T) is the relative amount of light that passes through a sample unabsorbed. Percent transmittance is easiest to measure, but absorbance (Abs = the amount of light actually absorbed) is a more convenient and useful parameter because it is directly and linearly proportional to the concentration of the absorbing substance (Figure 3). Absorbance can be calculated from transmittance as:

Figure 3. Absorbance (A) but not % Transmittance (B) is directly proportional to solute concentration.

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Using the Beer-Lambert Equation

The absorption coefficient (a) is characteristic of a particular substance at a specific wavelength and under a specific set of conditions, such as pH, solvent, etc. The relationship between a, concentration (c), and absorbance is known as the Beer-Lambert Law: Abs = (a) x (b) x (c) where b equals the path length (the distance the light traverses through the sample), which is usually 1 centimeter for most cuvettes. You may have seen the equation written as Abs = ,bc; where , is the molar extinction coefficient. We use a here because the concentration units of proteins are expressed as :g/:L or mg/mL, not in molarity. When using the absorption coefficient to determine concentration, the equation can be rearranged to solve for c c = Abs ) (a x b), and, since b = 1: c = Abs ) a

Creating a standard curve to estimate the Absorption Coefficient

One way to determine the absorbance coefficient is to prepare a graph called a Standard Curve, which is a plot of the absorbances of solutions containing known concentrations of the substance (Figure 4). The slope (m) of the line (y = mx + b) estimates the absorbance coefficient for the measured substance and can then be used to calculate the concentration of an unknown from its absorbance as c = Abs ) m Figure 4. Example Standard Curve In this exercise we will determine protein concentration of a homogenate of the mouse liver that you resected earlier this semester. We will measure the amount of protein using the BCA reagent, which in the presence of protein turns a purple color the more protein present the deeper the color. 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. It doesnt matter which type of protein is used to create the standard curve because the BCA reagent reacts the same with all protein, so you will use a protein called bovine serum albumin (BSA)

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The Absorbance Spectrum

Sometimes it is useful to examine the absorbance of a substance over a range of wavelengths to create an absorbance spectrum a plot of absorbance vs wavelength. Since different molecules have unique absorbance properties, absorbance spectrum can used to identify an unknown substance and determine its structural characteristics. An absorbance spectrum is often used to determine the wavelenth of light at which absorbance is greatest this is called the 8max (wavelength of maximum absorbance). Knowing the 8max of a substance is useful, since measurements at this wavelength will be Figure 5. Absorbance spectra of Bradford most sensitive. Figure 5 shows absorbance of the reagent in absence and presence of protein. Bradford reagent, which also can be used to measure protein concentration. Notice that the absorbance 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. In this lab exercise, you will prepare absorbance spectra for the BCA protein assay reagent and determine its 8max in the absence and presence of protein.

Light Scattering
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. Light scattering is a phenomenon that will cause an erroneously high absorbance measurement. Light scattering occurs when a sample is turbid (cloudy) due to the presence of suspended particles. As shown below in Figure 6, the particulates will deflect Figure 6. Effect of suspended particles on absorbance measurements 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.

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Using the Genesys 20 Spectrophotometer

The three essential steps for using the spectrophotometer are: A. select the wavelength B. insert the blank (control) sample and set the absorbance to 0'. C. insert and measure the absorbance of the test sample.

General Procedures 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 0. 4. Replace the sample with the test sample and record the absorbance.

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I. Homogenizing the liver and Determining the Protein Concentration.

In this part of the lab exercise you will be homogenizing the liver tissue and preparing it for analysis of protein content.

Supplies
13 mm test tubes ice bucket P-20 & P-200 pipeters 2 ml & 5 ml pipets

for homogenization: homogenization buffer sample of B-16 melanoma Potter-Elvejhem homogenizer for protein assay 25 ml BCA protein reagent spectrophotometer 2 cuvettes

15 ml centrifuge tube cryogenic storage vial

BSA protein standards heating block Figure 7. A BCA reagent standard curve is prepared by measuring the absorbance of samples with known amounts of BSA.

Procedures

All samples, solutions, and utensils must be kept chilled in ice at all times, unless indicated otherwise.

A. Homogenize the liver sample The amount of homogenization buffer used should be based upon the weight of your liver sample: 5 ml per gram., thus the volume needed will be weight of liver x 5 ml ______ g liver (your sample x 5 ml = ______ ml homogenization buffer. 1. Thaw the liver sample that you resected earlier this semester, and transfer it to a Potter Elvjem homogenizer. 2. Add the appropriate amount of homogenization buffer. 3. Homogenize with several passes of the pestle.

B. Centrifuge the homogenate 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. After the centrifugation, carefully transfer the supernatant to a labeled cryostorage vial held in an ice bucket.

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C. Measure the protein concentration of the homogenate You will prepare a "standard curve" of bovine serum albumin (BSA) vs its absorbance in the BCA reagent. You will use this standard curve to determine the concentration of your liver homogenate. Figure 7 shows again how the standard curve is made. 1. Dilute a portion of your homogenate 100x with homogenization buffer to a final volume of 5 ml (5000 :l). Measure the buffer with a 5 ml pipet and the homogenate with a P-200 pipetter. Keep this diluted sample on ice. Do the dilution in a 13 mm test tube. 5000 :l of a 100x dilution = _____ of homogenate + _____ of buffer Dilution is necessary to keep the protein concentration within an acceptable range for the BCA reagent. ***The BCA assay must be performed for all of the samples at the same time.*** *** The BCA reagent should not be chilled use at room temperature.*** 2. Label the standard curve and homogenate assay tubes. Label the 6 tubes to receive the BSA standard protein 0", 0.2", 0.4", 0.6", 0.8", and 1.0" :g/ml. Label the 4 tubes to receive sample homogenate Blank, #1", #2", #3". 3. Add the BSA standard curve assay samples to the 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/ml. Thaw and thoroughly mix these samples before using. To prepare the standard curve 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. 4. Add the tissue homogenate samples to 13 mm tubes. For your diluted liver homogenate, you will need to prepare 3 replicates and a blank. Add 100 :L of the 25X diluted sample to each test tube labeled #1, #2, and #3. Add 100 :L of homogenization buffer to the tube labeled blank. 5. Add 2 ml of the BCA reagent to each 13 mm test tube carefully but expediently, and incubate all samples at 45oC for 15 minutes. Use a 5 ml pipet to add the BCA reagent. 6. After allowing the tubes to cool 5 - 10 minutes, measure absorbance at 562 nm and record the data in Tables 1 and 2. Zero the spectrophotometer before measuring the absorbance of the BSA and liver samples. The sample containing 0 :g/ml BSA 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 samples from the standard curve for Part II *** D. Store your liver homogenate for later use in the - 80o freezer 1. Label the cryogenic storage vial with your name, date and tissue. 2. Chill the vial in your ice bucket, and then transfer your homogenate to the vial with a Pasteur pipet. 3. Place your vial in the designated container in the -80EC freezer. Spectrophotometry & Protein Measurement Page 7

Complete these calculations after lab

F. Determine the absorption coefficient for the BCA reagent used with BSA. Graph the standard curve (including 0 :g) using Excel (select the set intercept = 0" under Trendline Options). Absorbance coefficient = slope of trendline = __________ :L cm-1 :g-1 E. Calculate the protein concentration in the homogenates. Since c = Abs ) (a x b), and b = 1 therefore, c = Abs ) a, you can use the absorption coefficient to determine the protein concentration for the three liver replicas and then average the values. Calculate the protein concentration in the original homogenate (:g/:L), bearing in mind that the homogeneate was diluted 25X before measuring the absorbance. Avg. :g/:l protein x dilution factor = Protein concentration ______ :g X _______ = ______ :g/:L protein in the homogenate

II. Determine 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 (8max), and compare this to the 8max 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 dH2O 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 dH2O blank. Record your absorbance measurements in Table 3. 3. To narrow the wavelength at which the maximum absorbance (8max) occurs for the BCA + protein sample, take measurements at 5 nm intervals in the region of the 8max. Record your absorbance measurements in 4. 4. Plot the full absorbance spectra (420 - 700 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 8max. Spectrophotometry & Protein Measurement Page 8

Table 1. Data for protein assay standard curve.

Tube # 0 (blank) :g/:L BSA 0 Place complete table in lab notebook 5 1.0 Abs @ 562 nm

Graph the standard curve (including 0 :g) using Excel, selecting the set intercept = 0" under trendline options. Use the equation of the line to determine the protein concentration of the three liver replicas. Table 2. Data for liver homogenate.
Sample # (blank) 1 Place complete table in lab notebook 3 AVG: Abs @ 562 nm 0 :g/:L protein determined from standard curve Tara to 0

Avg. :g protein x dilution factor = Protein concentration in the homogenate ______ :g X _______ = ______ :g/:L protein The absorption coefficient is represented by the slope of the trendline Absorption coefficient = slope of trendline = __________ :L cm-1 :g For the upcoming electrophoresis lab exercise, you will need to work with a volume of your homogenate that contains 100 :g of protein; calculate this volume now: 100 :g ) Protein concentration in the homogenate = volume of homogenate

100 :g ) ______ :g/:L protein = __________ :L

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Table 3. Absorbance measurements for BCA only absorption spectrum.

Wavelength 420 nm Place complete table in lab notebook 700 Absorbance BCA only Absorbance BCA + BSA

Table 4. Absorbance measurements in 5nm increments around the 8max

Wavelength Absorbance BCA + BSA

Place complete table in lab notebook

QUESTIONS (Turn in typed answers.) Your answers to these question demonstrate how well you understand the principles of this lab exercise. Be sure to use ciorrect terminology 1. What are the functions of BCA and BSA in the protein assay procedure. Why is it necessary to perform the standard curve and the test samples at the same time? 2. Why is it necessary to centrifuge the homogenate before performing the protein assay? Why is it necessary to dilute the tissue homogenate before performing the protein assay? 3. The BSA standards are prepared by first making a stock solution of 1mg/ml, and then making dilutions to yield each final concentration. When preparing a standard curve, a mistake when preparing the BSA stock solution will cause greater inaccuracy than if you mispipet 1 or 2 samples. Explain why. 4. Does the 8max determined for the BCA + protein sample agree reasonably well with value at which the BCA reagent is used? Why is a wavelength of 562 nm used for measuring protein concentration instead of some other wavelength (explain why this wavelength will yield the most sensitive measurements)?

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