Lab #1.
Determination of Protein, RNA and DNA content of a Human Cell.
Author: Lee
Weber, Biology Dept, University of Nevada, Reno
Minor modifications by Grant Mastick
[Return
to BIO 395 Schedule]
I. Overview:
HeLa cells are a human tumor cell line originally derived from a cervical carcinoma over 30 years ago. They grow very well in vitro, either in suspension or in surface culture. You will observe HeLa cells under the compound microscope and determine the concentration of cells (per milliliter, ml) using a hemocytometer. The cells will then be collected by centrifugation and lysed in a measured volume of a detergent that dissolves cell membranes and denatures proteins. The concentration of DNA and protein in aliquots of the crude lysate will be measured with specific dye binding assays, which use fluorometric and colorimetric techniques. Total nucleic acid (DNA + RNA) will be isolated from the remaining lysate and quantitated by UV absorbance spectroscopy. You will then calculate the mass of total protein, RNA and DNA contained in a single cell.
II. Background:
A. Spectrophotometry: The wavelengths of light absorbed by a particular compound are determined by its molecular and electronic configuration. The particular absorbance characteristics are thus a fundamental physical property of that compound. The principle that governs which specific wavelengths are absorbed is that the energy of a photon is directly related to its frequency by Plank's constant. Since electrons, and to a lesser degree molecular bonds, can exist only at certain discrete energy levels, only photons that contain the energy required to increase the energy of the system to the next higher level will be absorbed. The absorption spectrum of a compound can be a unique characteristic, and is frequently used in the identification of the complex macromolecules. Many different types of spectrophotometers are used for spectral analysis. A spectrophotometer does not directly measure the amount of light absorbed by a sample, but rather the amount of light at any given wavelength that passes through (is not absorbed by) a sample. The fraction of light transmitted through an absorbant sample relative to a blank sample containing only solvent is the percent transmission or %T.
The absorbance (O.D.) of a sample is related to %T in the following way:
abs = 2 - log %T.
Spectrophotometers use light sources that emit in the ultraviolet, visible, or infrared region of the spectrum. The emitted light is fractionated into its component wavelengths by either a prism or a grating; a series of narrow slits are positioned such that the light is refined into a monochromatic band. The mechanical part of the instrument that performs this function is called a monochrometer. The monochrometer directs light of the desired wavelength through the sample. A photodetection system composed of one or two photomultiplier tubes is positioned on the opposite side of the sample. This proportionally converts light energy to an electrical output which can be read by a meter which is essentially a potentiometer. The instrument is first standardized by adjusting the output of a blank solution containing only solvent to equal 100% transmittance vs. the voltage output when the slits are blocked (0% transmittance). The concentration of a light absorbing compound in the same solvent is inversely related to the decrease in the % of light transmitted. However, the relationship is logarithmic. For this reason, all modern spectrophotometers convert % transmittance (%T) to absorbance (A) electronically by an inverse log function. A simple linear relationship exists between the amount of monochromatic light absorbed by a compound at its maximum extinction wavelength and its concentration.
The mathematical relationship between the concentration of a substance and
the absorption of monochromatic light is provided by the Beer-Lambert law. The
basis of this law comes from the work of Lambert on the transmission of
monochromatic light by homogeneous solid substances. Beer applied the law to
solutions and found that both the concentration and thickness (light path
distance) of the solution affect the transmission of light through it.
|
Lambert's Law |
log ( Io / I ) = k L |
where Io is the original intensity of the light beam; I is the
intensity of the beam after passing through the sample; L is the thickness of
the sample in cm; and k is the proportionality constant which depends on the
absorption characteristics of the sample under investigation.
|
Beer's Law |
log ( Io / I ) = k c |
where Io, I, and k are similar to Lambert's law and c is the
concentration of the sample in moles per liter.
|
Beer-Lambert Law |
log ( Io / I ) = k c L |
The log function is called the optical density (O.D.) or absorbance, k is the molar extinction coefficient and may be written as E. E is specific for a given wavelength and is the O.D. of a 1.0 molar solution of the substance, which can be very large at wavelengths that are absorbed efficiently.
The relationship between absorbance and concentration is:
|
Optical density |
O.D. = E c x 1 |
since the standard light path distance for most sample cuvettes is 1 cm.
The concentration (in moles per liter) of a substance in solution can be
calculated from the optical density, if no interfering compounds are present,
by solving for c:
|
Concentration |
c = OD / E |
In Molecular Biology, it is often not convenient to refer to the
concentration of macromolecules in molar terms. The concentration of DNA, RNA
and protein, molecular species that are heterogeneous in size, are usually
calculated in terms of mass per unit volume (gm/liter or mg/ml). If the
extinction coefficient of a 1 gm/liter (1mg/ml) concentration of a pure
substance is known, any concentration of that substance can be calculated from
optical density as follows:
|
Concentration |
c = OD / E1mg/ml |
The laboratory instructor will demonstrate the use of the spectrophotometer. Remember, it is always necessary to prepare at least two sample cuvettes: one is used to blank the instrument and must contain all of the components of the sample tube (solvents, buffers, substrates, etc.) except the substance for which the concentration is to be determined. This is inserted into the sample compartment and the instrument is adjusted to read 100% transmittance or 0.0 O.D. The other cuvette with the unknown sample is then substituted and the absorbance reading is obtained. In certain dual beam instruments such as the Beckman GT, the blank sample is left in place when the reading of the unknown is taken. The spectrophotometer then measures the difference in the intensity of the light transmitted by both the sample and the blank simultaneously.
B. Colorimetric Analysis: The major limitation of direct spectrophotometric measurement of cellular constituents is the presence of compounds that absorb light at similar wavelengths. For example, the concentration of a pure sample of protein can be determined by measuring absorbance of UV light at 280 nm, the wavelength at which most proteins absorbs most strongly. In a crude cell homogenate or lysate however, there are many other compounds that also absorb at this wavelength such as RNA, DNA, nucleotides, free amino acids and certain coenzymes and lipids. To circumvent this problem, specific colorimetric assays have been developed to measure individual macromolecular species. In general, these assays involve the reaction of the molecule with a dye of other compound that undergoes a spectral change in response to binding to or reaction with the molecular species to be measured. The change in absorbance of the indicator compound, which undergoes the color change, is then measured spectrophotometrically.
C. Fluorometry: The absorption of light energy by molecular bonds is a transient phenomenon. As the energy level of "excited" electrons returns to the ground state, the energy of the absorbed photons is released either in the form of molecular motion (heat) or emitted as photons of light at wavelengths different than those absorbed by the compound. Compounds that emit light are said to be fluorescent. The concentration of fluorescent compounds can be measured using a special type of spectrophotometric instrument called a fluorometer. Fluorescent compounds usually emit a characteristic spectrum of light when illuminated with a particular wavelength. For fluorometric measurement of concentration, a solution containing the compound of interest is illuminated with light at wavelengths close to the excitation maximum. Then the amount of light released at wavelengths at or near the emission maximum is measured by the instrument. Fluorescent dyes are used for fluorometric quantitation of specific compounds in a manor analogous to the use of light absorbing dyes in colorimetry. If the fluorescence spectrum of a dye is altered when it interacts with a specific target molecule, the concentration of the target molecule can be measured indirectly. As in colorimetric measurements, indirect fluorescence measurements are only accurate over a limited range of target compound to dye ratios, usually with the dye in excess.
III.
Procedure:
(Reminder: keep detailed notes in your notebook!)
A. Determination of Cell Number, Preparation of the Cell Lysate, and Isolation of the Nucleic Acid Fraction.
1. Obtain a 5-10 ml sample of a suspension culture of HeLa cells in a graduated centrifuge tube from your instructor. Write down the volume of your sample. Keep the cells on ice. (Please note that it is possible that we will be using another cell line if HeLa cells are not available.)
2. Suspend the cells by vortexing briefly and place 10 ul of the culture on a clean microscope slide using the P-20. Place a coverslip over the drop and examine the cells under the microscope using high power. Diagram a typical HeLa cell in your notebook. Indicate the cellular structures that you can distinguish with your own eyes. Be objective and do not use your imagination.
3. Measure the concentration of cells per ml with a hemocytometer as directed by your instructor. Indicate the number of cells counted, the cells/ml, and the total number of cells in the sample you were given. Show all of your calculations!
4. Collect your cells by centrifugation in a clinical centrifuge as directed by your instructor. Decant the tissue culture medium into a beaker and resuspend the cell pellet by vortexing in 1 ml of physiological saline solution. Transfer the suspension to a 1.5 ml microcentrifuge tube and collect the cells by centrifugation for 10 sec at full speed in the microcentrifuge. Immediately decant the supernatant and remove the last drop of saline with the P-20. Be very careful not to resuspend the cell pellet.
5. Add 396 ul of 10X TE buffer (0.1 M Tris, pH 8.0, 0.01 M EDTA) to the tube and thoroughly suspend the cells by vortexing. Lyse the cells by adding 4 ul of 10% sodium dodecyl sulfate (SDS) solution and vortexing vigorously. The solution should become somewhat viscous as high molecular weight DNA is released from the nucleus. Vortexing will shear the DNA and reduce the viscosity. Continue vortexing until the solution can be easily pipetted.
6. Remove 1/10 of the cell lysate (40 ul) to a fresh microfuge tube and hold on ice. This sample will be used for DNA and protein determination.
7. Total nucleic acid will be isolated from the remaining 9/10 (360 ul) of cell lysate. Add 40 ul of 10% SDS to the remaining lysate and vortex thoroughly at room temperature. Add 4 ul of a 10 mg/ml solution of proteinase K. (Proteinase K is a powerful proteolytic enzyme that is active in the presence of SDS. It will rapidly degrade the protein in the cell lysate to amino acids and small oligopeptides that are soluble in ethanol.) Mix by vortexing and incubate your sample for 15-30 min in a 37o bath.
8. When digestion is complete, add 200 ul of 7.5 M ammonium acetate solution to your sample, mix, and then add 1.2 ml of 100% ethanol. Mix by inverting the tube. Note the fibrous precipitate that forms. This is DNA. RNA also precipitates in ethanol/ammonium acetate, but it forms a finer suspension that is not always visible until collected by centrifugation. Allow the suspension to precipitate for 2 min at room temperature and then centrifuge for 10 min at maximum speed in the microcentrifuge.
9. Decant the ethanol supernatant, which contains nearly all of cellular proteins (as amino acids), the proteinase K, nucleotides, coenzymes, lipid, and other UV absorbing components of the cell. The pellet contains all of the DNA and RNA in a fairly pure form. Remove the last drop of ethanol with the P-20, being careful not to disturb the pellet.
10. Dissolve the pellet in 1.0 ml of 0.1 N NaOH for determination of
absorbance at 260 nm as described below.
B. Measurement of Protein Concentration.
The protein concentration of present in the cell lysate will be determined
using the method of Bradford, as we did in the first day of class. The Bradford
procedure is a dye-binding assay based on the differential color change of
Coomassie blue G dye as it binds to protein. In both research and clinical
applications, this assay has replaced earlier procedures for protein
measurement (ie. the Lowery method) because of its simplicity, the stability of
the color change, and the lack of interference from non-protein components. The
Bradford assay is based on the observation that the absorbance maximum for an
acidic solution of the dye shifts from 465 nm to 595 nm when binding to protein
occurs. Beer's Law may be applied for accurate quantitation of protein in
solution by selecting an appropriate ratio of dye volume to sample
concentration. Within a range of 5-100 ug of protein, a linear relationship
exists between protein concentration and the increase in absorbance of the dye
solution at 595 mn. Over a broader range of protein concentration, the dye
binding method gives an accurate, but not entirely linear response.
Carry out the same procedure as your group did for the first
day of class. First make a protein standard curve using BSA as
provided. Then pipette 10 ul of your cell lysate into 90 uL of distilled
H2O in a microcentrifuge tube. Vortex well, then use 20 uL of the diluted
cell lysate for the determination of protein concentration.
Calculate the amount of protein in 10ul of lysate by using
the standard curve. Then calculate the amount of protein in the entire lysate.
If the absorbence of your sample is greater than the most concentrated sample
in the standard curve, dilute a sample of your lysate with H2O
following the directions of your lab instructor and repeat the assay. Using the
absorbance value of a dilution that is within the linear range of the standard
curve, calculate the protein concentration of the undiluted lysate and the
total amount of protein in the lysate. If your first dilution is not
within the linear range of the standard curve, make more or less dilute, and repeat
the measurement.
|
ug protein/10ul Total protein in lysate (400 ul) = --------------------- X 40 dilution factor (if necessary) |
(Do you understand what each component in this equation
is, and why it is there?)
C. Determination of DNA Concentration.
The fluorometric method of Labarca and Paigen will be used to measure the amount of DNA in your cell sample. The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoechst 33258) binds with DNA. You will be using a fluorescence detector that is specifically designed for this particular assay and so is much simpler to use than a conventional fluorometer. The instrument uses filters to produce an excitation wavelength at about 355 nm and detects light emitted at about 490 nm. Under the conditions used in the assay (2 M NaCl), DNA is the only cellular constituent that increases the emission of 490 nm light by Hoechst 33258. Over a range of DNA to dye ratios that has already been optimized, the increase in fluorescence is directly proportional to the amount of DNA.
1. Be sure that the fluorometer has been turned on and warmed up for at least 30 min. Obtain a solution of Hoechst 33258 at 0.1 ug/ml in TNE buffer (0.01 M Tris, pH 7.5, 2 M NaCl, 0.001 M EDTA) and a DNA standard solution at a concentration between 100 an 250 ug/ml (100 - 250 ng/ul). The dye is a potential carcinogen, so use gloves, and do not get it on your skin or clothes.
2. You have been provided with one glass cuvette. Make sure the cuvette is optically clean. Rinse the cuvette with distilled H2O and rinse the outside with ethanol. Dry with a Kimwipe. When handling the cuvette, take care not to touch any of the lower surfaces. Fingerprints will interfere with your readings. The cuvette should be rinsed thoroughly after each set of readings.
3. Pipette 2.0 ml of the working dye "blank" solution into the clean cuvette. Insert the cuvette into the measuring well, close the cover, turn the scale knob fully clockwise, and adjust the zero control knob so that the LCD meter reads 000; minor fluctuations are normal at this sensitivity setting.
4. Remove the cuvette, pipette in 2.0 ul of the DNA reference standard. Mix the contents of the cuvette by inverting 4-5 times after sealing the top with a piece of parafilm. Reinsert the cuvette and adjust the scale knob to set the LCD readout to match the concentration of the standard (e.g. if the standard contains 195 ng/ul, set the LCD to read 195).
5. Rinse the cuvette with dist. H2O and drain by inverting on a paper towel or kimwipe. Add a fresh 2.0 ml dye blank; check the 0 setting and adjust if necessary. Now add 2.0 ul of your cell lysate to the cuvette, mix and read the concentration of DNA/ul directly from the LCD meter (e.g. if your sample reads 50, the concentration of DNA in your lysate is 50 ng/ul).
6. Calculate the amount of DNA present in your entire 400 ul cell extract.
D. Measurement of Total Nucleic Acid and Calculation of Total RNA.
The nucleic acid sample you have extracted from 360 ul of lysate is pure enough to allow the direct spectrophotometric measurement of concentration by measuring the absorbance of the solution at 260 nm, the wavelength at which both DNA and RNA absorb maximally. By measuring absorbance in 0.1 N NaOH (pH 13), the effect of differences in secondary structure between DNA and RNA is eliminated as DNA is denatured to the single stranded form and RNA is unfolded and degraded to nucleotides. Under these conditions a 1 mg/ml solution of either RNA or DNA gives an absorbance of 20 at 260 nm. Since the amount of DNA has been measured independently by fluorometry, it will be possible to calculate the amount of RNA in the total nucleic acid sample by subtracting the known amount of DNA.
1. Measure the absorbance of your 1.0 ml total nucleic acid fraction at 260 nm in a spectrophotometer as described by your instructor. If the absorbance of your sample is greater than 1.0, dilute the sample 5 or 10 fold with 0.1 N NaOH and redetermine the absorbance. The same rules for handling cuvettes apply as for fluorometry.
2. Calculate the concentration of total nucleic acid in your sample.
|
Concentration (in mg/ml) |
c = O.D.260 / 20 |
3. Calculate the amount of total nucleic acid in the lysate. Remember that you only measured the nucleic acid in 360 ul of the lysate.
4. Calculate the amount of RNA in the entire lysate by subtracting the fraction of the mass contributed by DNA.
E. Final Calculation.
1. Using the data you obtained from the cell count, calculate the mass of protein, DNA, and RNA contained in a single HeLa cell.
References: links to Pubmed database
Bradford,
M., Anal. Biochem. 72:239 (1976)
Labarca,
C. and Paigen, K., Anal. Biochem. 102:344 (1980)