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How can you measure the concentration of proteins in a solution? It is difficult because you can't count the proteins directly. An indirect method, called spectrophotometry, is to shine a light through the solution and measure how much of it is blocked by the proteins. More precisely, you can bind a fluorescamine molecule to every protein molecule. The combination of the two bound molecules blocks a particular shade of violet light, wavelength 415 nm, that is not blocked by other materials in the solution. A spectrophotometer shines a known amount of such light on the solution and measures how much gets through. If more light is blocked, the concentration of protein in the solution is higher. You are going to use spectrophotometry to measure a protein concentration today.
Students will be able to:
Describe the theory underlying photospectrometry, including the selectivity of fluorescent labeling, and explain how it is used to measure protein concentrations.
Demonstrate the following practical laboratory skills:
Use R to create and plot a standard curve and perform a regression analysis.
Use a standard curve to determine an unknown protein concentration.
BEFORE CLASS, please read the following: NEEDS ADJUSTING TO NEW MATERIALS
CAN THE INTRO BE GREATLY SHORTENED WITHOUT LOSS TO THE LAB EXPERIENCE?
Would be better to show the excitation and emission spectrum for fluorescamine bound to protein than ethidium bromide bound to DNA since this lab uses fluorescamine.
One of the main challenges faced by a cell biologist in designing an experiment is being able to observe what is actually happening. Cells are complicated and tiny; not only is it difficult to see objects within the cell that are hundredths or thousandths of millimeters in size, but there are so many different cellular components in so small a volume that it is often difficult to pick out individual structures. Light microscopy can allow biologists to examine entities as small as organelles within a cell, yet there are many experimental questions that cannot be addressed by light microscopy. For example, the concentration of calcium ions within a cell is of great importance in countless biological pathways, yet no light microscope exists that can allow researchers to “see” calcium directly. Similarly, cell biologists often wish to determine the fate or location of a particular protein within a cell, yet it is difficult or impossible to identify a single protein from among the hundreds to thousands of other proteins that are present without some experimental manipulation. One strategy that researchers use to examine biological processes that cannot be studied using light microscopy involves related phenomena known as absorbance and fluorescence (specialized microscopes called “fluorescence microscopes” can be used to visualize these cell products and are discussed further below).
You will recall from your chemistry classes that molecules (including the molecules that are found in and make up cells) are comprised of atoms connected by shared electrons. Under normal conditions, each of these electrons exists at a low energy level known as its ground state. In addition to the ground state energy level, the electrons can also exist at higher energy levels, known as excited states. See Figure 1. Electrons can move back and forth between their ground and excited states: they can be promoted or excited from a low-energy state to a higher-energy state when they absorb a photon of light, and relax from a high-energy state to a lower-energy state when they give off, or emit, a photon of light.
FIGURE 1 HERE
Figure 1. (A) An electron is promoted from its ground state to its first excited state by absorbing a photon with the same energy as the energy difference between the two levels. (B) The electron relaxes back to its ground state by emitting a photon with the same energy as the energy difference between the two levels.
However, two quantum mechanical requirements complicate this picture.
First, the possible electron energy levels are discrete (that is, there are only a handful of possible energy levels for a given electron.) For example, if a particular electron’s ground state has an energy of 1 eV (an eV, or electron-Volt, is a unit of energy), and its lowest excited state has an energy of 2 eV, then the electron cannot have 1.5 eV of energy (or 1.04 eV, or 1.762 eV, or 1.348 eV….).
Second, in order for an electron to be promoted from one energy level to another, it has to absorb a photon of light with an energy corresponding to the change in the electron’s energy level. In other words, if an electron is excited from a 3 eV energy level to a 4.5 eV energy level, then it must absorb a photon with 1.5 eV of energy (= 4.5 eV – 3 eV). Similarly, if an electron relaxes from a 4.5 eV energy level to a 3 eV energy level, then a photon with 1.5 eV of energy is emitted.
Importantly, the energy of photons is inversely related to the wavelength of the corresponding light, denoted by λ: The longer the wavelength, the lower the energy. The wavelength, in turn, correlates to the light’s color. Humans can see wavelengths ranging from around 350-400 nm (purple) up to around 750-800 nm (red). Shorter wavelengths (< 350 nm) correspond to ultraviolet, gamma, and X-rays; longer wavelengths (> 800 nm) correspond to infrared, radio, and microwaves. See Figure 2.
FIGURE 2 HERE
Figure 2. The electromagnetic spectrum. Higher energy photons are at the left; lower energy photons are at the right. Note that shorter wavelengths correspond to higher energy photons.
Cell biologists can take advantage of the above properties of light and electrons using a technique known as spectrophotometry. A spectrophotometer is an instrument that measures the amount of light at a given wavelength absorbed by a solution. The following diagram illustrates the basic design of the spectrophotometer.
DIAGRAM OF SPECTROPHOTOMETER HERE
Figure number. Caption.
The spectrophotometer contains a source of white light (recall that white light consists of a spectrum of colored light with a range of wavelengths). The white light shines through a filter which allows only a narrow band of wavelengths to pass. Different filters can be selected to change the wavelength of analysis. The spectrophotometer shines the selected wavelength of light on the sample, and the intensity of light which passes through the sample is detected by a photoelectric sensor. The instrument then compares the intensity of light which entered the sample (the incident light) with the intensity of light exiting the sample (transmitted light). If the selected wavelength matches up with the gap between energy levels in the sample, then some of the light will be absorbed by the sample, and the intensity of transmitted light will be less than the intensity of the incident light. The result of this analysis is reported by the spectrophotometer as the optical density (OD) or absorbance. These experiments are most often used to measure absorbance at wavelengths ranging from 200-800 nm (the ultraviolet and visible portions of the spectrum), this type of experiment is known as UV-vis spectroscopy.
An important consideration is that when we perform a spectroscopy experiment on a solution, we are interested in the number of photons absorbed by the solute itself, not by the cuvette walls, plate, solvent, or air between the light source and phototube. For this reason, we always include a blank sample that is identical to our experimental samples, except that it does not contain our test solute. For example, if you were measuring the absorbance of the amino acid tyrosine in a phosphate buffer, your blank sample will consist of the same phosphate buffer in an identical cuvette, but no tyrosine. All spectrophotometers require measurement of a blank sample prior to measurement of your experimental sample; the instrument automatically subtracts the blank’s absorbance from the experimental sample’s absorbance. Because your blank sample and experimental sample differ only in the presence of your test solute, any absorbance that arises from the solvent, cuvette, etc., is not reflected in the value reported by the spectrophotometer, and the reported absorbance corresponds only to the solute’s absorbance.
UV-vis spectroscopy experiments have a useful property. It turns out that the absorbance of a given sample varies linearly with concentration (that is, increasing the concentration of a solute by a factor of three will increase the absorbance of the solution by a factor of three). Put in mathematical terms, we can express the relationship between absorbance and concentration as follows:
A = kc
where A is absorbance, c is concentration, and k is a constant. (An expanded version of this equation is known as Beer’s Law). The constant k depends on the particular solute being measured. In order to convert absorbance into concentration, we take advantage of the linear relationship between absorbance and concentration and measure a standard curve. A standard curve is generated by determining the absorbance readings for a set of solutions that contain known concentrations of the molecule of interest, and plotting them on a graph. We can then use this graph to determine the value of k and ultimately convert the absorbances of unknown solutions into concentrations.
Absorbance is a relatively straightforward process: a molecule absorbs a photon of a specific wavelength, promoting an electron from its ground state to an excited state. Measuring this absorbance provides a mechanism to determine the concentration of photon-absorbing molecules in solution. However, there is a phenomenon related to absorbance that has proven to be especially useful to cell biologists, known as fluorescence. In general, once an electron absorbs a photon of the appropriate wavelength and is promoted to a higher energy level, the excited electron eventually relaxes back to its ground state, emitting a photon with the same wavelength as the one it originally absorbed. However, imagine an electron that is excited by a photon from its ground state to its second excited state. In certain molecules, called fluorophores, the excited electron can actually relax down to the first excited state without emitting a photon. After this initial relaxation, the electron returns to its ground state, this time by emitting a photon. You’ll notice, however, that the energy level of the emitted photon is different (lower) than the energy level of the absorbed photon. Then, instead of looking for absorbance of photons, we look for emission of lower energy photons, as in Figure 3. It is possible to measure fluorescence much more sensitively than absorbance; whereas measuring absorbance requires measuring the slight dimming of a bright light, fluorescence spectroscopy looks for light that is produced directly by the sample. (Imagine the difference between spotting a lit match in a dark room, versus trying to tell the difference between a 100 W lightbulb shining in your eyes and a 75 W lightbulb shining in your eyes.)
FIGURE 3 HERE
Figure 3. Fluorescence occurs when a single molecule absorbs and emits photons of two different wavelengths. In the first step (left), a fluorophore absorbs a photon, promoting an electron from its ground state to its second excited state. The electron next relaxes down to its first excited state, often without emitting a photon (middle). Finally, the electron relaxes back to its ground state, emitting a photon with a lower energy and longer wavelength than the initially absorbed photon (right).
Besides allowing highly sensitive detection of fluorophores, an additional advantage of fluorescence experiments is that very few biological molecules are fluorescent, whereas many biological molecules absorb photons (for example, DNA and RNA absorb photons at 260 nm, and proteins absorb photons at 220, 254, and 280 nm). Thus, if we could specifically link a fluorophore to a biological molecule of interest, we should be able to detect it very specifically.
There are three main ways that fluorophores can be linked to molecules of interest.
Some proteins are naturally fluorescent; the most famous example of fluorescent proteins is GFP (“green fluorescent protein”), for which Roger Tsien, Osamu Shimomura, and Martin Chalfie won the Nobel Prize in Chemistry in 2008. It is relatively straightforward to genetically engineer a cell so that a single protein is linked to GFP.
Antibodies are a class of proteins that bind very specifically to foreign molecules, known as antigens. It is possible to generate antibodies that recognize nearly any molecule, as well as chemically link a fluorophore to the antibody. Then, by mixing the fluorescently labeled antibody with the experimental sample, the antibody will specifically bind to the molecule of interest.
The third common way to label a molecule with a fluorophore is by staining. This is the least specific means of labeling molecules within a cell; staining is most useful when you want to study a class of molecules instead of a particular molecule. For example, DAPI is a fluorophore that specifically binds to, or stains, DNA; if you are interested in labeling all the DNA within a cell, you could stain the cell with DAPI. Note that DAPI does not let you locate a specific DNA sequence, however. Similarly, ethidium bromide is another dye that stains nucleic acids and is the one you used to visualize the DNA in the gel electrophoresis experiment you performed earlier in the semester.
Once we have labeled a molecule with a fluorophore, how do we observe it? There are several experimental techniques that take advantage of fluorescence.
Use a fluorescence spectrometer
Similar to a UV-vis spectrometer, except it only detects the photons emitted by the fluorophore.
Use a fluorescence microscope
Conceptually similar to light microscopes, it allows observation of fluorophores within a cell. A fluorescence microscope shines light on the sample designed to excite its fluorophores; the optics in the microscope include an emission filter that only allows light of the wavelength of the fluorescently emitted photons to reach the eyepiece and the user. Fluorescence microscopes allow cell biologists to observe the locations of fluorescently labeled molecules within a cell with high specificity and sensitivity.
Use a flow cytometer.
Flow cytometers measure fluorescence of individual cells as they pass through the instrument and provide a readout of the number of fluorescent vs. nonfluorescent cells. Flow cytometers are a powerful tool in immunology.
Typically there is a range of wavelengths that can excite a particular fluorescent molecule and another range of wavelengths in which they emit but in both cases there is a peak. Ethidium bromide has a peak excitation wavelength of 300 nm and a peak emission wavelength of 595 nm. This explains why you use a UV illuminator to visualize bands on a DNA gel and why the bands appear orange. See Figure 4.
FIGURE 4 HERE
Figure 4. Fluorescence excitation (blue) and emission (red) of ethidium bromide bound to DNA. Taken from invitrogen.com website (Product Spectra-Ethidium Bromide) 26 Aug 2009. MORE PRECISE REFERENCE OR NEW SOURCE NEEDED. Can not find this figure at the website. Caption refers to colors not visible in lab manual.
For the purposes of this lab, it is logistically difficult to measure the fluorescent emission of photons from a fluorophore. Instead, you will use a spectrophotometer to measure the absorbance arising from the initial electron excitation of a fluorophore known as fluorescamine, a fluorescent stain specific for proteins. You will be provided with solutions of fluorescamine, DNA, and a protein known as bovine serum albumin (BSA). First, you will generate a series of BSA solutions with known concentrations. You will treat these solutions with fluorescamine; the fluorescamine will stain the BSA, and you will use these solutions to generate a standard curve for fluorescamine-stained BSA. Using this standard curve, you will determine the concentration of BSA in an unknown solution. Finally, to demonstrate the selectivity of fluorescence as an experimental technique, you will also treat a sample of DNA with fluorescamine and measure its absorbance. If fluorescamine is indeed specific for proteins, you should not see any absorbance in the stained DNA solution.
EQUIPMENT LIST IS INCOMPLETE
Do volume calculations for making 16 solutions used in building a standard curve.
You will be combining BSA stock solution, buffer stock solution, and water. The requirements for your solutions are:
| Cfinal (BSA) | Vstock (BSA) | Vstock (Na phosphate) | Vwater | Vfinal |
|---|---|---|---|---|
| 0.00 mg/mL | 0.00 µL | 20 µL | 180 µL | 200 µL |
| 0.05 mg/mL | 200 µL | |||
| 0.10 mg/mL | 200 µL | |||
| 0.15 mg/mL | 200 µL | |||
| 0.20 mg/mL | 200 µL | |||
| 0.25 mg/mL | 200 µL | |||
| 0.30 mg/mL | 200 µL | |||
| 0.35 mg/mL | 200 µL | |||
| 0.40 mg/mL | 200 µL | |||
| 0.45 mg/mL | 200 µL | |||
| 0.50 mg/mL | 200 µL | |||
| 0.60 mg/mL | 200 µL | |||
| 0.70 mg/mL | 200 µL | |||
| 0.80 mg/mL | 200 µL | |||
| 0.90 mg/mL | 200 µL | |||
| 1.00 mg/mL | 200 µL |
A. Calculate the volume of buffer (Na phosphate) needed for each solution and enter it in the Dilutions Table. The buffer stock solution is 1 M.
B. Calculate the volume of BSA stock solution needed for each solution and enter it in your table. The BSA stock solution is 2 mg/mL.
You can use the dilution equation to calculate the volume of BSA stock solution needed:
Dilution Equation: Cstock x Vstock = Cfinal x Vfinal
where
C. Calculate the volume of water needed for each solution and enter it in your table.
Make 16 solutions to be used for building the standard curve.
For each row of the table, combine Vstock(BSA), Vstock(Na phosphate), and V(H20) in an Eppendorf tube. Each tube will contain 200 µL of solution.
Make one additional 1.0 mg/ML BSA solution as in the bottom row of the Dilutions Table, and put it in an Eppendorf tube. It will not be labeled.
Get 2 samples from your instructor.
Label the BSA and DNA solutions with fluorescamine.
Set aside one of the 1.0 mg/mL BSA tubes, leaving it unlabeled.
Add 67 µL of 1.08 mM fluorescamine in acetone to all the other tubes.
Mix gently by flicking the tube several times, trying to avoid introducing bubbles.
Wait five minutes, then transfer 200 µL of each fluorescamine-treated solution to separate wells of a 96-well plate.
Use the plate reader to measure the absorbance of your samples at 415 nm.
Please complete the following activities. When answering questions, please be thorough in your explanation. For numerical answers, always show your calculations. You may work on the questions collaboratively, but if you do, you must be very clear on this by submitting one set of answers with an Honesty Agreement. Everyone must submit an individual and unique graph for Question 1.
Working individually, create a Google spreadsheet of your group’s data and generate a graph that shows a standard curve. Include all important features of a complete graph, not forgetting the figure legend.
Did you see any absorbance at 415 nm in the tube to which you did not add fluorescamine? How is this relevant to fluorescamine’s usefulness as a stain?
Did you see any absorbance at 415 nm in your DNA only sample? Why does this make fluorescamine a useful reagent for cell biologists?
The Unknown sample you have been given is __________.
What is the concentration of DNA in your unknown sample?
Propose an alternative strategy to determine the concentration of DNA in your unknown sample.
Fluorescamine is not fluorescent except when it is attached to a protein. Why was this property necessary for the experiment you performed today?
The BSA stock solution was made from powder. One day you might have to make it yourself.
The molecular weight of BSA is 69323 g/mol.