BIOL 212 Lab 3

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California State University, Long Beach *

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212

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Dec 6, 2023

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Fall 2018 3-1 PROTEIN QUANTIFICATION OF CELLULAR FRACTIONS .......... 3 INTRODUCTION Last week crude homogenate and fractions of nuclei, mitochondria and cytoplasm were collected from cauliflower inflorescence tissue. These fractions contain cellular protein as well as other cellular components. Although the same volume (milliliters) of each fraction was collected, the concentration of protein in each fraction will vary. Today, the concentration of protein in each fraction will be measured using a dye (Coomassie Brilliant Blue G-250) that binds to arginine, histidine and aromatic amino acids (phenylalanine, tryptophan and tyrosine). Under acidic conditions, dye binding to amino acids results in a shift in the absorbance maximum from 465 nm to 595 nm, which is measured by spectroscopy at 595 nm. Reaction schematic for the Coomassie Brilliant Blue G-250 dye. To assign a specific concentration of protein to a specific A 595 value (where A is absorbance), a standard curve will be made using known concentrations of bovine serum albumin (BSA), an abundant protein isolated from cow blood plasma. Four different concentrations of each fraction as well as crude homogenate will be assayed at the same time as the BSA samples. A 595 values will be determined, and the BSA samples will be used to produce a standard curve. This standard curve will be used to assign a protein concentration ( P g/ml) to the A 595 values obtained from your crude homogenate, nuclei, mitochondria and cytoplasm samples. MATERIALS BSA solution (50 P g/mL), Tube Series #1 of cellular fractions (from exercise 2), Coomassie Brilliant Blue G-250, distilled water, 0.1% SDS in 1 mM EDTA,

Fall 2018 3-2 spectrophotometer tubes, spectrophotometer, large glass test tubes, water bath (boiling), rulers, permanent marking pens, goggles A. DETERMINATION OF λ max FOR THE BSA PROTEIN/DYE COMPLEX CAUTION: WEAR THE PROVIDED GOGGLES AND GLOVES DURING THIS EXPERIMENT. THE COOMASSIE DYE WILL STAIN YOUR SKIN AND CAN CAUSE PERMANENT VISION LOSS. COOMASSIE BLUE SOLUTION IS STRONGLY ACIDIC. 1. The wavelength of maximum absorption ( λ max ) for the BSA protein/Coomassie dye complex will be verified experimentally. This is the specific wavelength at which the absorbance of radiant energy by the complex is the strongest. Begin by obtaining two spectrophotometer tubes and labeling them A and B. Into tube A, pipette 0.5 mL of water and into tube B pipette 0.5 mL of the 50 μ g/mL BSA stock solution. Pipette 2.0 mL of Coomassie reagent into each tube. 2. Mix each tube by covering the top with a small square of Parafilm and inverting each three times. Allow the tubes to sit at room temperature for five minutes. 3. Measure the absorbance of tube B at wavelengths spanning from 545 to 645 nm at 10 nm intervals using tube A to zero the spectrophotometer. Be sure to re-zero the spectrophotometer each time the wavelength is changed. Record the data you obtain in Table 3.1. Using this data, construct an absorption spectrum by plotting absorbance on the y-axis versus wavelength on the x-axis. At what wavelength does the λ max occur?

Fall 2018 3-3 Table 3.1 Absorbance of the BSA Protein/Coomassie Dye Complex at Various Wavelengths Wavelength (nm) Absorbance of Tube B 545 555 565 575 585 595 605 615 625 635 645 B. PREPARATION OF STANDARD CURVE USING BSA 1. Prepare a dilution series for the protein BSA using the provided 50 μ g/mL stock solution. Begin by labeling three glass spectrophotometer tubes 2-4 and then Tubes A and B from part A can be re-labeled as tubes 1 and 5 respectively and re- read with tubes 2-4 after preparation. In order from left to right in each column, add the indicated volumes of the stock BSA solution and distilled water as shown in Table 3.2, which will generate the new BSA concentrations shown. Then add the Coomassie Blue Reagent and mix each tube by covering the top of the tube with a small square of Parafilm and inverting it three times. Allow the tubes to sit at room temperature for five minutes prior to reading any of their absorbance values. 2. Set the spectrophotometer to 595 nm, the wavelength of maximum absorption ( O max ) for the protein/Coomassie Blue reagent complex. Use tube #1 (which was Tube A in part A) as a blank to zero the spectrophotometer. To measure the absorbance of tubes 2-5, begin by inverting each tube three times (covered with

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Fall 2018 3-4 Parafilm), wiping its surface with a Kimwipe and placing it into the chamber of the spectrophotometer. Record your data in Table 3.2. 3. Plot the results obtained during step 2 on the graph paper provided, plotting the absorbance for each tube on the y-axis, and the protein concentration (with the units of μ g/ml) on the x -axis. This will be your standard curve . TABLE 3.2. Absorbance at λ max as a Function of BSA Concentration Tube # BSA Stock Solution (ml) Distilled Water (ml) Coomassie Reagent (ml) Absorbance at 595 nm BSA Concentration ( μ g/ml) 1 (A) 0 0.5 2 0 2 0.1 0.4 2 10 3 0.2 0.3 2 20 4 0.3 0.2 2 30 5 (B) 0.5 0 2 50 C. CALCULATION OF PROTEIN CONTENT OF CELLULAR FRACTIONS (Refer to the flow chart in figure 3.1 also) 1. A dilution series of each of the four fractions will be prepared in order to find a protein concentration that lies within the linear range of the standard curve you prepared in part B. Please obtain 28 spectrophotometric tubes before proceeding. You are provided with 4 large test tubes for the 1:5 dilutions. The remaining dilutions will be done in spectrophotometric tubes. You will be using Tube Series #1. Recall that Tube Series #1 was obtained during the Cellular Fractionation experiment. Pipette 400 μ L from each cellular fraction into four separate large glass test tubes. Add 1.6 mL of 0.1% SDS in 1 mM EDTA and place each in a large non-spectrophotometric tube in the boiling water bath for two minutes (or 5 min if we are using the bath armor beads). This will lyse all of the membranes surrounding the organelles. Remove the tubes with the tongs provided . USE CAUTION, THE STEAM MAY CAUSE BURNS TO THE SKIN . These samples are a 1:5 dilution of the original fractions (Follow Figure 3.1 and Table 3.3). 2. In four clean spectrophotometer tubes, combine 0.4 mL of each of your 1:5 dilutions with 1.6 mL of distilled water. Mix the contents of these tubes thoroughly. This second set of samples is your 1:25 dilutions (Follow Figure 3.1 and Table 3.3).

Fall 2018 3-5 3. In four additional clean spectrophotometer tubes, combine 0.2 mL of each of your 1:5 dilutions with 1.8 mL of distilled water. Mix the contents of these tubes thoroughly. This third set of samples is your 1:50 dilutions (Follow Figure 3.1 and Table 3.3). 4. In four additional clean spectrophotometer tubes, combine 0.2 mL of each of your 1:25 dilutions with 1.8 mL of distilled water. Mix the contents of these tubes thoroughly. This third set of samples is your 1:250 dilutions . At this point, you should have four tubes for each of the four fractions for a total of 16 tubes. 5. Now determine the protein content in each of the 16 samples. Obtain 16 clean spectrophotometer tubes and label them 2-17 (use the same tube as the blank as you did in parts A and B, re-zeroing should not be necessary). Pipette 0.5 mL of water and 2.0 mL of the Coomassie reagent into the first tube. Transfer 0.5 mL of each sample into the remaining 16 tubes and add 2.0 mL of Coomassie reagent. Mix each tube after the dye is added by covering the top of the tube with a small square of Parafilm and inverting it three times. Allow the tubes to sit at room temperature for five minutes prior to reading any of their absorbance values. Measure the absorbance at 595 nm for tubes 1-17 (tube 1 or tube A as it was initially label should read 0 since it was the blank for part B). Record the data in Table 3.4. This assay is temperature dependant. Make sure your samples are at room temperature before reading. 6. Using the standard curve you created, determine the concentration of protein ( μ g/ml) in the 16 diluted samples. Record your calculated concentrations in Table 3.4. From these values, determine the actual protein concentration of the undiluted fractions in the units of μ g/ml (This is done once you have your diluted concentrations and multiply by them by the dilution factor of 5, 25, 50 or 250). Record these values also in table 3.4. Note however that any absorbance values cannot be used if they are greater than your highest absorbance reading on your standard curve. In this case, write N.A. for those sample dilutions (original and diluted) in Table 3.4.

Fall 2018 3-6 Figure 3.1 Flow Chart for Part C (dilutions of cellular fractions prior to estimation of protein concentration) (Total of 28 spec. tubes required and 4 large non-spectrophotometric tubes) Use 4 large tubes provided on your bench trays (1 for each series 1 fraction): 400 P L of each sample + 1.6 mL of 0.1% SDS-EDTA solution, heat for 2 minutes if using hot water or 5 min if using bead armor in the heating units This is the 1:5 dilution Use 4 small spec tubes Use 4 spec tubes 400 P L of each 1:5 sample + 1.6 mL dH 2 O 200 P L of each 1:5 sample + 1.8 mL dH 2 O 1:25 dilution (1/5 *1/5 =1/25) 1:50 dilution (1/5* 1/10=1/50) Use 4 small spec tubes 200 P L of each 1:25 sample + 1.8 mL dH 2 O 1:250 dilution (1:25 *1/10=1/250) For each of the 16 tubes above (1/5, 1/25, 1/50 and 1/250 dilutions): Transfer 0.5 mL sample into a new tube (label with fraction name and dilution) + 2 mL Coomassie blue Top with parafilm, mix, wait 5 minutes. Record absorbance values after zeroing with tube #1 in Table 3.4

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Fall 2018 3-7 TABLE 3.3. Set up your test tubes as shown below to facilitate making the dilutions of your cellular fractions. Dilution Crude Homogenate Nuclear Mitochondrial Cytosolic 1:5 2 6 10 14 1:25 3 7 11 15 1:50 4 8 12 16 1:250 5 9 13 17 As shown by arrow C, the 1:5 dilutions are used to make the 1:50 dilutions (200 P L of 1:5 and 1.8 mL of water). As shown by arrow A the 1:5 dilutions (400 P l of 1:5 and 1.6 mL water) are also used to make the 1:25 dilutions. The 1:250 dilutions are made (follow arrow B) by taking 200 P L of 1:25 dilution and addition 1.8 mL of water. TABLE 3.4. Protein Concentration Determination for Cellular Fractions Tube # Dilutions of Fractions (From Tube Series #1) Absorbance at 595 nm Protein Concentration Diluted sample ( P g/ml) Original fraction ( P g/ml) Crude Homogenate 2 1:5 3 1:25 4 1:50 5 1:250 Nuclear 6 1:5 7 1:25 8 1:50 9 1:250 Mitochondrial 10 1:5 11 1:25 12 1:50 13 1:250 Cytosolic 14 1:5 15 1:25 16 1:50 17 1:250 A B C

Fall 2018 3-8

Fall 2018 3-9

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Fall 2018 3-10 Questions 1. In general terms, describe how the Coomassie dye-binding assay works for detection of proteins. 2. How was a lambda maximum (wavelength maximum) empirically determined? Did your maximum coincide with the published maximum? 3. A standard curve with known concentrations of BSA was made. Which cellular fraction dilutions in Table 3.4 were you able to obtain a value for concentration using your plotted standard curve? 4. Using the Beer-Lambert Law (A= e c l), determine the concentration of a protein solution that has an absorbance value of 0.5 and a extinction coefficient of 0.016 P M - 1 cm -1 at the O max of 400 nm. (Please look at the equation on page 3-5 closely, and assume that l = 1 cm.) 5. As observed from Table 3.4, if you were to compare the 1/50 dilutions of each cellular fraction, are all original protein concentrations in P g/mL the same? If you were to pipette 0.5 mL of each of these 1/50 dilutions, what would be the amount of protein in P g that you would have pipetted? Complete the table below comparing fractions at this dilution and protein content in P g. A 595 , 1/50 dilution [protein], μ g/ml μ g of protein in 0.5 ml Crude Homogenate Nuclear Mitochondria Cytoplasm (A= e c l) ,

Fall 2018 3-11 6. List your four samples from above in order of most to least concentrated and re- write your protein concentrations from question 5 here. Is this consistent with what you would expect when you think about a eukaryotic cell? Think about the number of each type of organelle and the protein content of cellular compartments. Fraction name μ g/ml Most concentrated: 2 nd most concentrated: 3 rd most concentrated: Least concentrated: 7. Calculate the micromolar ( μ M) extinction coefficient (e; units are μ M -1 cm -1 ) for BSA protein assuming that it has a molecular weight of 66,000 g/mole. The final units should be P M -1 cm -1 . This extinction coefficient is the slope of your standard curve. Convert the slope units of ml/ P g to P M -1 cm -1 Portions of the protocols used in this lab were adapted from the following source: Mason, A. Z., Carlberg, D., Gharakhanian, E., Brusslan, J., and Palmier, C. 2004. Laboratory Manual For Biological Sciences I , 7 th ed. Pearson Custom Publishing.