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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
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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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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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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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
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
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
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
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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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