Exercise 1

Determination of DNA and Protein Concentration of Bovine Liver Tissue

Exercise 1- 1

INTRODUCTION All cells contain an enormous variety of organic compounds. One important goal of molecular biology is to identify and quantify a cell's contents, since the amount and type of certain molecules may provide clues as to the processes occurring inside the cell. Of all the compounds found in cells, the nucleic acids and proteins could be considered the most important (or at least the most interesting), since they are responsible for information storage and usage. This exercise is designed to demonstrate two approaches to the measurement of DNA and protein in cells of bovine liver tissue. Generally it is not possible to measure the amount of DNA or protein directly, without extensive purification. Instead we use an indirect method that will work with relatively impure samples. One technique uses the ability of each type of compound to undergo a specific chemical reaction producing a colored end-product whose concentration can be measured with a spectrophotometer. The second uses the inherent ability of many molecules to absorb specific wavelengths of electromagnetic energy, in this case ultraviolet (UV) light. Visible Light Spectrophotometry (Colorimetry) Diphenylamine assay. This colorimetric assay is used to measure the concentration of DNA in cells or tissues (Schneider, 1957). The reagents used in the diphenylamine reaction include acetic acid and sulfuric acid. When these are heated with DNA they cleave the phospho-diester bonds and hydrolyze the glycosidic bonds between the sugar and purines. The free 2-deoxy ribose undergoes a dehydration reaction to form -hydroxylevulinyl aldehyde, which reacts with diphenylamine to produce a variety of blue-colored compounds showing a characteristic absorbance peak at 595 nm. The more DNA there is in the sample, the darker the blue color will be. Since the reaction is specific for 2-deoxy ribose, the sugar in DNA, there is no reaction with the ribose sugar of RNA. Thus, the presence of RNA in a sample will not interfere with the measurement of DNA. For colorimetric reactions such as this, you must establish a standard curve by measuring the absorbance (at a specific wavelength) of a series of known concentrations of DNA . You will then compare the absorbance obtained from your cell sample to the standard curve to estimate the concentration of DNA in the sample.
Exercise 1- 2

BioRad (Bradford) protein assay. This is a commercial assay system which uses a blue dye (Coomassie blue) that binds specifically to proteins (Sedmak and Grossberg, 1977). The details of the process are not well understood, but it seems probable that the dye reacts with the free amino groups present on the basic amino acids. It is a moderately sensitive test that is fast and cheap. For more accurate, sensitive and reliable protein assays most scientists use a method known as the 'Lowry test,' but this is more complex and time consuming. Like the diphenylamine reaction, the BioRad assay involves a colorimetric reaction. You will need to make a standard curve, using purified bovine plasma albumin as the standard protein. Use extreme caution when working with the BioRad reagent as it contains phosphoric acid and methanol, and it will stain your skin. Ultraviolet (UV) Spectrophotometry This technique makes use of the fact that nucleic acids absorb ultraviolet light very strongly at a wavelength of 260 nm, while proteins absorb ultraviolet most strongly around 280 nm, to detect the presence of these molecules in a sample. Although UV absorption can be used to measure the quantity of nucleic acids (or proteins) in a sample, it cannot distinguish between DNA and RNA. Furthermore, considerable purification of the sample is required before the technique can be used for quantitative analysis.

Exercise 1- 3

Background reading Read through the entire laboratory handout before you come to lab. In order to understand the laboratory procedures and their rationale, you will need some background information about nucleic acids and the methods to be used. Much of this information is presented in this handout; the rest will come from your lecture notes, your textbook, or from other reference sources. You should also become familiar with the theory (i.e., Lambert-Beer laws) behind the use of a spectrophotometer to analyze organic molecules (e.g., Cooper, 1977). You will be using the Bausch and Lomb Spectronic 20 spectrophotometer for quantifying the color changes in both the diphenylamine and BioRad reactions, so review the procedures for setting the wavelength and zeroing this instrument. Be sure that you understand what a standard curve is, how it is produced, and how it is used. Also, make sure that you understand the relationship between absorbance (measured in optical density units) and % transmittance. *** Why do we make readings in absorbance rather than % transmittance for most spectrophotometric work? *** For spectrophotometric work requiring ultraviolet (UV) wavelengths, a more sophisticated spectrophotometer that can emit and detect UV light will be used. The procedures for operating of this instrument will be explained by your instructor.
REFERENCES Cooper, T.G. (1977) The Tools of Biochemistry. Wiley-Interscience, JohnWiley and Sons, New York. Elser, J.J., D.R. Dobberfuhl, N.A. MacKay,and J.H. Schampel (1996) Organism size, life history, and N:P stoichiometry. BioScience 46(9): 674-684. Schneider, W.C. (1957) Determination of nucleic acids in tissues by pentose analysis. In: Methods of Enzymology, Vol. 3, Colowick, S.P. and Kaplan, N.O., eds., pp. 680-684. Academic Press, New York. Sedmak, J.J. and S.E. Grossberg (1977) A rapid, sensitive, and versatile assay for protein using Coomassie Brilliant Blue G-250. Analyt. Biochem. 79: 544-552. Exercise 1- 4

PROCEDURES:
Each group should have one member working on the DNA extraction and one on the extraction of soluble proteins. The remaining members of the group should prepare the reaction tubes for the diphenylamine and BioRad assays. Extraction of nucleic acids and soluble proteins: 1. Weigh out 60 g of frozen beef liver and cut it into small pieces. 2. Place 200 ml ice-cold water into a pre-chilled blender. Turn the blender on to high speed, then slowly add the pieces of liver. Homogenize for 1-2 minutes; the homogenate should have a uniform appearance with no tissue chunks. Filter approximately 100 ml of the homogenate through 6 layers of cheesecloth. 3 Add 15 ml of this filtrate to each of four 30 ml centrifuge tubes, balance the tubes, and spin at 10,000 x g for 30 minutes at 4C. Proceed to step 11. This material will be used for determination of protein concentration. Note: TCA is extremely corrosive. Use extreme caution when handling. Clean up spills with plenty of water. 4 The remaining unfiltered homogenate will be used for DNA extraction. A member of each group should place 4 mls of the homogenate into a chilled 40 ml glass centrifuge tube and add 5 mls of ice-cold 10% trichloracetic acid (TCA) 5. Mix the TCA and liver cell homogenate thoroughly by drawing up and down in a Pasteur pipette. This step will precipitate all macromolecules, including nucleic acids, proteins, and lipids.

Exercise 1- 5

6. Centrifuge the suspension at 1000 x g for 2 minutes at 4C in the bench-top centrifuge and discard (decant) the supernatant. Wash the pellet by adding another 5 ml of cold 10% TCA and repeating the mixing and centrifugation. 7. Discard the supernatant. Add 10 ml of 95% ethanol. Break up any clumps using a pipette and centrifuge for 2 minutes at 1000 x g. This step extracts lipids, which are soluble in ethanol. 8. Discard the supernatant, and add 10 ml of 5% TCA and resuspend the pellet. Place the tube in a 90C water bath for 15 minutes. During this time, remove the tube every few minutes and gently agitate. This step will degrade nucleic acids, mainly to soluble nucleotides, but leaves proteins largely intact (although denatured) in the pellet. 9. Centrifuge at 1000 x g for 2 minutes. DO NOT THROW AWAY THE SUPERNATANT OR THE PELLET. Transfer the supernatant to a large test tube. 10. Add 10 ml of 5% TCA to the pellet, mix well and centrifuge at 1000 x g for 2 minutes. Add the supernatant to that from #9. Measure the DNA concentration of this combined supernatant (= liver TCA extract) using the diphenylamine assay. ****************************** 11. The samples from step 3 should be removed from the centrifuge and the supernatant carefully decanted into a small beaker. Dilute 1 ml of supernatant with 19 ml water (= 1:20 dilution) and mix. Make a second dilution by mixing 1 ml of supernatant with 99 ml water (= 1:100 dilution). Measure the protein concentration of the two dilutions of supernatant using the BioRad assay.

Exercise 1- 6

Diphenylamine assay for DNA: 1. Prepare six cuvettes according to the table below: a blank tube (to zero the spectrophotometer), four tubes containing known amounts of DNA, and a tube with your sample of the liver TCA extract. The DNA stock is already prepared, and contains 400 g DNA/ml. Tube Blank 1 2 3 4 5 DNA Stock 0.0 ml 0.5 ml 1.0 ml 1.5 ml 2.0 ml 0.0 ml Liver TCA extract 0.0 ml 0.0 ml 0.0 ml 0.0 ml 0.0 ml 2.0 ml 5% TCA 2.0 ml 1.5 ml 1.0 ml 0.5 ml 0.0 ml 0.0 ml

Note: diphenylamine reagent contains sulfuric and acetic acids. Use extreme caution when handling. Clean up spills with plenty of water. 2. When all the tubes are ready (including tube 5 containing the liver extract), add 4 ml of the diphenylamine reagent to each tube. Cover with Parafilm and invert twice, to mix the contents. 3. Remove the Parafilm, cap the tubes with foil, wrap with tape, and label each tube using a pencil. Place the tubes in a boiling water bath for 10 minutes (DO NOT SUBMERGE TUBES IN WATER!). Remove the tubes from the boiling water and cool them briefly in an ice bath, then allow them to reach room temperature. Once the tubes are dry, read their absorbance at 600 nm. 4. Set the spectrophotometer to 600 nm, zero it using the blank tube, then determine the absorbance of the four DNA standards and your sample of liver extract (tube 5). ***What is the final DNA concentration of each of the DNA standards?***

Exercise 1- 7

5. Construct a standard curve by plotting absorbance (y-axis) vs DNA concentration (x-axis) for the standards and draw the best-fitting line through the points. Use this standard curve to estimate the DNA concentration in the liver extract. Calculate the DNA concentration (e.g., g DNA/g tissue) in the original liver tissue.

Bradford assay for Protein: 1. Prepare 4 cuvettes, each containing 0.1 ml of one of the purified albumin standards (0, 150, 600, 1200 g protein/ml) plus 5 mls of the BioRad assay reagent. Cover each tube with Parafilm and invert several times. 2. Prepare two liver extract assay tubes by adding 0.1 ml of the diluted liver extract supernatants (1:20; 1:100) to separate cuvettes and adding 5.0 mls BioRad reagent to each. Cover with Parafilm and mix well. 3. Set the Spec 20 to 595 nm. Using the 0 g/ml albumin standard as your blank, zero the instrument and obtain the absorbance readings for each of the standard tubes and for the liver extract tube. 4. Construct a standard curve and estimate the protein concentration in the liver extract. Calculate the protein concentration (eg. g protein/g tissue) in the original sample of liver tissue. Ultraviolet Absorption Spectra 1. If time permits, ultraviolet absorption spectra over the wavelength range 230-320 nm should be generated using samples of purified calf thymus DNA (dissolved in 5% TCA) and purified bovine albumin (dissolved in distilled water). Your instructor will explain the operation of the UV spectrophotometer. 2. If time permits, UV absorption spectra should also be generated for the aqueous supernatant containing soluble proteins (used for the BioRad assay), and for the TCA supernatant containing the extracted nucleic acids (used for the diphenylamine assay).
Exercise 1- 8

Data Analysis While writing your lab report, you should think about the following questions and try to answer them in the discussion section: 1. What are the advantages and disadvantages of using the colorimetric assays to estimate the amount of DNA and protein in tissue? 2. What are the concentrations of DNA and soluble protein per gram of beef liver tissue? How much DNA and protein is there per cell? Do these values ever change within a cell? Would these values be the same in a different type of tissue from the cow? Would these values be the same in liver cells from a different species? You should consider the following questions, even if your lab group did not measure the UV absorption spectra: What are the advantages and disadvantages of using the UV absorption spectra to estimate the amount of DNA and protein in the sample of liver tissue? How do you explain any differences in the UV absorption spectra for purified DNA and protein? What kinds of information do these spectra provide? What should the absorption spectra for the aqueous protein extract and the TCA nucleic acid extract look like?

June 2005 MA

Exercise 1- 9