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
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
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 0. 4. 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
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
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
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 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 8 max. Protein Measurement and Absorption Spectrophotometry Page 6
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
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