Lecture Notes for Methods in Cell Biology

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Lecture Notes for Methods in Cell Biology (TRMD 623) Instructor: Mark F. Wiser 1 TABLE OF CONTENTS PREFACE—COURSE DESCRIPTION ................................................................................7 CHAPTER 1MICROSCOPY ..............................................................................................9 LIGHT MICROSCOPY .............................................................................................9 ELECTRON MICROSCOPY...................................................................................12 MICROSCOPY APPENDIX. ALDEHYDE FIXATIVES ......................................16 CHAPTER 2SPECTROPHOTOMETRY..........................................................................19 SPECTROPHOTOMETRY THEORY ....................................................................19 INSTRUMENTATION ............................................................................................21 APPLICATIONS OF SPECTROPHOTOMETRY..................................................21 FACTORS AFFECTING ABSORPTION ...............................................................22 VARIATIONS IN SPECTROPHOTOMETRY.......................................................22 APPENDIX I. PHOTOMULTIPLIER TUBE..........................................................24 APPENDIX II. CALCULATION OF ENZYME ACTIVITY.................................25 CHAPTER 3FLUORESCENCE........................................................................................29 ENZYME ASSAYS AND FLUOROMETERS.......................................................29 FLUORESCENCE MICROSCOPY.........................................................................30 FLOW CYTOMETRY .............................................................................................33 CHAPTER 4RADIOCHEMISTRY ...................................................................................35 ATOMIC STRUCTURE AND RADIOACTIVE DECAY......................................35 PROPERTIES OF RADIOISOTOPES ....................................................................36 MEASUREMENT OF ß-RADIATION ...................................................................37 GAMMA-RAY DETECTION .................................................................................42 SAFETY ...................................................................................................................42 APPENDIX 1. INTERNAL STANDARDS.............................................................43 APPENDIX 2. CHANNELS RATIO .......................................................................44 APPENDIX 3. MULTIPLE ISOTOPES ..................................................................45 CHAPTER 5pH AND BUFFERS......................................................................................47 DEFINITIONS .........................................................................................................47 HENDERSON-HASSELBACH...............................................................................47 INSTRUMENTATION ............................................................................................48 COMPLICATIONS..................................................................................................48 BUFFERING CAPACITY .......................................................................................49 BUFFER SELECTION ............................................................................................49 BUFFER APPENDIX ..............................................................................................50 2 CHAPTER 6CENTRIFUGATION....................................................................................53 SEDIMENTATION THEORY.................................................................................53 PREPARATIVE CENTRIFUGATION ...................................................................55 CENTRIFUGATION APPENDIX 1. RCF CALCULATION.................................58 CHAPTER 7INTRODUCTION TO PROTEINS..............................................................61 PROTEIN STRUCTURE .........................................................................................61 PROTEIN STABILITY............................................................................................63 PROTEIN ASSAYS .................................................................................................64 APPENDIX. AMINO ACIDS: PROPERTIES AND STRUCTURE ......................66 CHAPTER 8DIFFERENTIAL PRECIPITATION OF PROTEINS..................................69 PROCEDURE FOR (NH ) SO PRECIPITATION.................................................69 4 2 4 OTHER PROTEIN PRECIPITATION METHODS ................................................70 APPENDIX. AMMONIUM SULFATE NOMOGRAM .........................................71 CHAPTER 9CHROMATOGRAPHY ...............................................................................73 BASIC PRINCIPALS...............................................................................................73 EQUIPMENT ...........................................................................................................74 ADSORPTION CHROMATOGRAPHY.................................................................75 ION EXCHANGE CHROMATOGRAPHY ............................................................75 HYDROPHOBIC CHROMATOGRAPHY .............................................................79 Polar Solvent.........................................................................................................................80 GEL FILTRATION CHROMATOGRAPHY..........................................................80 AFFINITY CHROMATOGRAPHY........................................................................81 CHAPTER 10MEMBRANES AND DETERGENTS.......................................................83 TYPES OF MEMBRANE PROTEINS....................................................................84 DETERGENTS.........................................................................................................85 DETERGENT REMOVAL ......................................................................................87 APPENDIX 1. CELL DISRUPTION TECHNIQUES.............................................88 CHAPTER 11ELECTROPHORESIS................................................................................91 GEL ELECTROPHORESIS.....................................................................................91 EQUIPMENT ...........................................................................................................93 SDS-PAGE ...............................................................................................................94 ISOELECTRIC FOCUSING....................................................................................96 TWO-DIMENSIONAL GEL ELECTROPHORESIS..............................................97 PROTEIN DETECTION FOLLOWING ELECTROPHORESIS............................98 PREPARATIVE ELECTROPHORESIS ...............................................................101 ELECTROPHORESIS APPENDIX 1. SIZE CALCULATION............................104 3 ELECTROPHORESIS APPENDIX 2. GEL STAINING ......................................105 CHAPTER 12PROTEIN PURIFICATION OVERVIEW...............................................107 MICROSEQUENCING AND PEPTIDE MAPPING ............................................110 APPENDIX. FOLD PURIFICATION AND RECOVERY ...................................113 CHAPTER 13IMMUNIZATION ....................................................................................115 IMMUNOGENICITY ............................................................................................115 IMMUNOGEN PREPARATION...........................................................................116 IMMUNIZATION..................................................................................................117 COLLECTING AND PROCESSING BLOOD .....................................................120 ANTIBODY PURIFICATION...............................................................................120 CHAPTER 14MONOCLONAL ANTIBODIES .............................................................123 IMMUNIZATION..................................................................................................123 FUSION..................................................................................................................124 SELECTION...........................................................................................................124 SCREENING..........................................................................................................125 CLONING ..............................................................................................................125 PRODUCTION.......................................................................................................126 APPENDIX. B-CELL DIFFERENTIATION ........................................................127 CHAPTER 15IMMUNOASSAYS...................................................................................128 IMMUNOPRECIPITATION .................................................................................129 IMMUNOBLOTTING ...........................................................................................131 IMMUNOFLUORESCENCE ................................................................................133 IMMUNOGOLD ELECTRON MICROSCOPY ...................................................134 ELISA (AND RIA).................................................................................................135 DIRECT VS. INDIRECT .......................................................................................136 OTHER METHODS...............................................................................................138 CHAPTER 16NUCLEIC ACID STRUCTURE AND ISOLATION...............................141 ISOLATION OF NUCLEIC ACIDS......................................................................142 ISOLATION OF RNA ...........................................................................................145 DENSITY GRADIENT CENTRIFUGATION......................................................146 ANALYSIS AND QUANTIFICATION................................................................146 APPENDIX 1. COMMON CONVERSIONS ........................................................147 CHAPTER 17MODIFICATION OF NUCLEIC ACIDS ................................................149 RESTRICTION ENDONUCLEASES ...................................................................149 4 CHAPTER 18ELECTROPHORESIS OF NUCLEIC ACIDS.........................................153 PULSE FIELD GEL ELECTROPHORESIS .........................................................155 CHAPTER 19HYBRIDIZATION AND BLOTTING TECHNIQUES............................157 GENERAL PROCEDURES...................................................................................157 FACTORS AFFECTING HYBRIDIZATION.......................................................159 PREPARATION OF LABELED DNA PROBES..................................................160 RFLP AND RESTRICTION MAPPING ...............................................................163 IN SITU HYBRIDIZATION..................................................................................164 DNA MICROARRAYS .........................................................................................165 CHAPTER 20POLYMERASE CHAIN REACTION......................................................167 PCR MECHANISM ...............................................................................................167 PRACTICAL ASPECTS ........................................................................................168 RNA-PCR...............................................................................................................170 QUANTITATIVE PCR ..........................................................................................170 LIMITATIONS.......................................................................................................172 PRECAUTIONS.....................................................................................................174 CHAPTER 21RECOMBINANT DNA ............................................................................175 PREPARATION OF FOREIGN DNA...................................................................175 PLASMID VECTORS............................................................................................178 BACTERIOPHAGE λ............................................................................................184 FILAMENTOUS BACTERIOPHAGE..................................................................188 COSMIDS AND YACS .........................................................................................190 QUALITY CONTROL...........................................................................................191 CHAPTER 22EXPRESSION OF RECOMBINANT PROTEINS...................................193 EXPRESSION IN E. COLI. ...................................................................................193 PREPARATION OF EXPRESSION VECTORS. .................................................195 SCREENING λ LIBRARIES WITH ANTIBODIES.............................................195 EXPRESSION IN EUKARYOTES .......................................................................196 APPENDIX 1. CODES AND CODONS ...............................................................198 APPENDIX 2. FUSION PROTEINS .....................................................................199 APPENDIX 3. GENERATING FRAMESHIFTS..................................................200 CHAPTER 23DNA SEQUENCING ................................................................................201 DIDEOXY CHAIN TERMINATION....................................................................201 EXTENDED SEQUENCING STRATEGIES........................................................204 MAXIM AND GILBERT.......................................................................................207 CHAPTER 24SEQUENCE ANALYSIS AND BIOINFORMATICS ............................208 5 SEQUENCE ALIGNMENT...................................................................................208 SEARCHING DATABASES.................................................................................210 PROTEOMICS.......................................................................................................211 APPENDIX 1. WEBSITES.....................................................................................216 APPENDIX 2. RESULTS OF BLAST SEARCH...................................................217 6 PREFACE—COURSE DESCRIPTION This course provides students with a broad overview to the basic biochemical, molecular and immunological techniques that are commonly used in modern biomedical research. Lectures will describe the theories and principals behind each of the methods in addition to discussing the practical aspects and limitations in executing the various procedures. One of the course objectives is to assist students with their own research by providing them with sufficient background information so that they are able to design experiments and know which methods are best suited to address a particular research question or problem. A second course objective is to provide students a better access to the scientific literature in that a better understanding of the methods will allow the students to critically evaluate the results and conclusions of scientific papers. Students anticipating careers involving biological or medical research at any level will benefit from this course. The course consists of three sections: 1) general biochemical and biophysical methods, 2) analysis and isolation of proteins and immunological procedures, and 3) analysis of nucleic acids and recombinant DNA. The first section will cover some basic biochemical procedures and equipment. Understanding these basic biochemical principals will assist in the subsequent discussions on proteins and nucleic acids. The section on characterization of proteins will describe some basic methods used to analyze proteins and provide an overview on protein purification. In addition, the generation of antibodies and their uses in various assays will also be covered. The final section on nucleic acids will describe the basic procedures used in molecular biology including gene cloning, PCR and sequence analysis. These lecture notes approximately follow the course and are divided into four sections: 1) General Biochemical and Biophysical Methods (Chapters 1-6), 2) Analysis and Characterization of Proteins (Chapters 7-12), 3) Immunological Methods (Chapters 13-15), and 4) Nucleic Acids and Recombinant DNA (Chapters 16-23). Many of the chapters correspond to the lectures. 7 PART I General Biochemical and Biophysical Methods Topics covered: • Microscopy • Spectrophotometry • Fluorescence and Flow Cytometry • Radioactivity • pH and Buffers • Centrifugation 8 CHAPTER 1MICROSCOPY Cells are small and in almost all situations a microscope is needed to observe them and their subcellular components. In fact the invention of the microscope led to the discovery and description of cells by Hooke in 1655. The microscope is still an extremely important tool in biological research. The light microscope has a limited capability in regards to the size of a particle that can be examined. The electron microscope provides additional resolution that allows for the examination of subcellular structures and even molecules. LIGHT MICROSCOPY The principal of light microsco- py is to shine light through a specimen and examine it under magnification. The major optical parts of a microscope are the objective lens, the eyepiece, the condenser and the light source. The objective lens functions to magnify the object. The high degree of magnifica- tion of the objective lens results in a small focal length and the magnified image actually appears directly behind the objective. The eyepiece functions to deliver this image to the eye or camera. Eyepieces also magnify the image, but it is an empty magnification. In other Major components of a light microscope words, the eyepiece enlarges the image but does not increase the ability to see fine details (i.e., the resolution). The condenser functions to focus the light source on the specimen. The condensor also eliminates stray light and provides an uniform illumination. An iris diaphragm which controls the amount of light reaching the specimen is also associated with the condenser lens. In addition, the light intensity can also be controlled by adjusting the voltage applied to the lamp on some microscopes. 1. Center light source Before using a microscope (Box) it is also important to check and all components that all of the optical components are centered on an optical axis on optic axis. so that the best image and resolution are obtained. Aligning the 2. Focus objective. optical components is usually simple (see instructions manual 3. Focus condenser. for the particular microscope) and needs to be done periodically. 4. Adjust illumination. The specimen is then placed on the stage and the objective lens is focused. The quality of the image produced is highly dependent on the illumination. The position of the condenser lens is adjusted so that the light is focused on the specimen and the intensity of the illumination is adjusted. On better microscopes the illumination can be controlled by both adjusting the diameter of the iris and by adjusting the voltage applied to the lamp. The amount of illumination is important for controlling resolution vs. the contrast and the depth of field (see Box for definitions). Resolution and contrast are antagonistic in that improving one results in a loss of the other. Resolution is increased by 9 increasing the amount of light. However, the brighter light leads to a loss in contrast. The user must decide upon the optimal mix of contrast and resolution by adjusting both the voltage (i.e., intensity or brightness) of the lamp and the iris diaphragm. The iris diaphragm also has some effect on the depth of field. Resolution The ability to discern fine details. Typically expressed as a linear dimension describing the smallest distance needed between 2 objects so that both are seen. Contrast Contrast refers to the number of shades in a specimen. More shades decreases the contrast, but increases the amount of information (also called dynamic range). Depth of Field Refers to the thickness of the specimen that will be in acceptable focus. Sample Preparation Specimens can be examined by simply placing them on a glass microscope slide under a glass cover slip. However, it is usually necessary to prepare and stain the samples before examination by microscopy. Fixation is a process by which cells are preserved and stabilized. Common fixatives include: acids, organic solvents, formaldehyde and glutaraldehyde (see Appendix for more discussion about aldehyde fixatives). These treatments affix macromole- cules in position. For example, glutaraldehyde chemically cross-links the primary amines of neighboring proteins and organic solvents precipitate proteins and other macromolecules. Thick samples, such as tissues, will need to cut into thin sections. Following fixation the sample or cells are embedded into a supporting medium. Paraffin is a common embedding medium for light microscopy as well as various plastic resins. Sectioning is carried out with a microtome (Figure). The microtome cuts the specimen into thin slices, or sections, of a specified thickness. It is also possible to collect the successive slices, called serial sections, and therefore ascertain the three dimensional aspects of the tissue or specimen being examined. The image generated by microscopy depends upon different components in the sample interacting with and impeding the light waves differentially. Biological samples are fairly homogeneous (i.e., carbon-based polymers) and do not greatly impede light. Therefore, it is often necessary to stain cells with dyes to provide more contrast. Different dyes have different affinities for different subcellular components. For example, many dyes specifically interact with nucleic acids (i.e., DNA and RNA) and will differentially stain the cytoplasm and nucleus. These stained subcellular components will differentially absorb the light waves and result in less light reaching the eyes or camera, and thus appears darker. Furthermore, since the dyes only absorb certain wavelengths of light, the 10 various structures within the specimen will exhibit • Dark Field different colors. (See chapter on Spectrophotometry for a • Phase Contrast more extensive discussion of chromaphores and light • Differential Interference absorption.) Contrast (or Normarski) • Confocal Scanning Variations to bright field (transmission) microscopy • Fluorescence • Image Enhancement Many modifications of light microscopy that have specialized applications have been developed (Box). In dark-field microscopy the specimen is illuminated from the side and only scattered light enters the objective lens which results in bright objects against dark background. This is accomplished through the use of an annular aperture that will produce a hollow cone of light that does not enter the objective lens (see Figure). Some of the light hitting objects within the specimen will be diffracted into the objective lens (see Figure Inset). The images produced by dark-field microscopy are low resolution and details cannot be seen. Dark-field microscopy is especially useful for visualization of small particles such as bacteria. Dark Field Microscopy Optics Phase Shift vs Diffraction Both phase contrast microscopy and differential-interference-contrast allow objects that differ slightly in refractive index or thickness to be distinguished within unstained or living cells. Differences in the thickness or refractive index within the specimen result in a differential retardation of light which shifts the phase or deviates the direction of the light (Figure). During phase contrast microscopy the phase differences are converted to intensity differences by special objectives and condensers. Normarski optics use special condensers and objectives to recombine incident and diffracted light waves from a single source at the plane of the image. In both methods the interference effects between the incident and diffracted light enhance small differences in the refractive index or thickness of the specimen and leads to an increased resolution without staining. In fluorescence microscopy a fluorochrome is excited with ultraviolet light and the resulting visible fluorescence is viewed. This produces a bright image in a dark background. 11 Confocal microscopy uses the objective lens as both the objective and the condenser. This allows the illuminating light to be focused on a relatively thin plane. In addition, a ‘pin-hole’ is used to further minimize the light coming from other planes. Minimizing the interference from other planes increases apparent resolution. (Fluorescence and confocal microscopy are discussed in greater detail in the chapter on Fluorescence.) Video cameras and image processing have had a major impact on microscopy. Images are digitized and can be manipulated electronically. This can correct imperfections in optical systems and can overcome limitations of human eye. In particular, the human eye is not very effective in dim light and cannot distinguish small differences in intensity against a bright background. Image enhancement can remedy both of these limitations. However, image enhancement cannot increase the resolution. This is due in part to the limit of resolution which is determined by the wavelength (λ) of visible light (see Box). The theoretical limit of resolution is defined as 0.61λ/N.A., where N.A. (numerical aperture) is a property of the objective lens determined by its magnification, diameter and refractive index. Typical ranges for the N.A. are 0.25-1.32. Visible light has an average wavelength of approximately 0.5 µm making the maximum limit of resolution approximately 0.2 µm. Mitochondria are about the smallest subcellular structures that theoretically can be seen. No amount of refinement of the optical systems can overcome this physical barrier, even though the image can be enlarged indefinitely. In addition, the practical limit of resolution will be less than the theoretical limit of resolution due to optical aberrations in the lenses (see Table). Typical Limits of Resolution for Common Objective Lenses ELECTRON MICROSCOPY Objective Theoretica Practical Magnificatio The relationship between the limit of n N.A. l (µm) (µm) resolution and the wavelength of the illumina- 4X 0.10 3.05 3.4 tion holds true for any form of radiation. Thus 10X 0.25 1.22 1.3 resolution can be increased in theory by using 40X 0.65 0.47 0.52 radiation of lower wavelengths. However, the 100X 1.30 0.24 0.26 human eye is only capable of detecting radiation with wavelengths in the range of 0.4-0.8 µm, or the visible spectrum. These problems associated with the limit of resolution have been overcome by using electrons to generate an image of the specimen. Particles, such as electrons, travelling near the speed of light behave as a wave (i.e., radiation) and their effective wavelength is inversely proportional to electron's velocity. Therefore increased resolution can be achieved by examining a specimen with high velocity electrons. The general principal of electron microscopy is analogous to light microscopy (Figure) except that electrons are used to analyze the specimen instead of visible light. The illumination source is a white-hot tungsten filament, which emits high velocity electrons. The electron beam is focused by a condenser lens onto the specimen. The condenser lens, however, is an electromagnet instead of a glass. These electrons are differentially impeded by the various struc- tures within the specimen. In other words, some of the electrons are scattered or absorbed by the atoms of the specimen. The electrons which pass through the specimen are focused with a series 12 of magnetic objective lens onto either a photographic plate or a fluorescent screen. The electrons interact with the photographic plate or fluorescent screen as if they were photons (i.e., light) and generate an image. The differential loss of electrons due to the substractive action of the sample will generate an image in much the same way as the absorption of light creates an image in light microscopy. Comparison of Microscope Optics Sample preparation 1. Fixation 2. Dehydration It is not possible to view living material with an electron 3. Embedding microscope. Biological samples are usually fixed with glutaralde- 4. Sectioning hyde, which cross-links proteins (see Appendix), and treated with 5. Staining osmium tetroxide, which stabilizes lipid bilayers and proteins. Osmium tetroxide is reduced by many organic compounds, especial- ly lipids, which results in cross-linking. Since electrons have very little penetrating power, samples must be embedded in special plastic resins and cut into thin sections of 0.05-0.1 µm. Removing all water from the specimen is necessary for the proper polymerization of the plastic resin. Following fixation the samples are dehydrated by exposing them to series of increasing alcohol concentrations until reaching 100%. The dehydrated sample is then put into a solution containing monomers of the embedding resin and polymerization is induced. This 'block' containing the sample is sectioned with the ultramicrotome and the ultrathin sections are place onto copper or nickel grids coated with a thin carbon or plastic film for support. Contrast in electron microscopy is dependent upon atomic number of the atoms in 13 the sample. Biological materials, primarily made of carbon, exhibit low atomic number and exhibit a similar electron scattering as the carbon films on the support grid. To obtain more contrast, samples are stained with salts of heavy metals, such as osmium, uranium and lead. Staining can be carried out before the dehydration and embedding or after sectioning. Different cellular compartments and structures stain differently with the heavy metals. Electron microscopes are expensive instruments and require substantial training to operate. Electron microscopy will usually require collaboration with someone having expertise in electron microscopy. Many universities have shared instrument facilities in which users pay a fee that includes use of the instrument and technical assistance. Typically fixed samples are provided to the electron microscopy service for further processing and analysis. Variations in Electron Microscopy • Transmission (TEM) • Scanning (SEM) The standard form of electron microscopy involves • Shadow-casting shooting an electron beam through the sample. This is called • Freeze-fracture transmission electron microscopy, often abbreviated TEM. • Freeze-etching Scanning electron microscopy (SEM) detects the electrons • Negative staining that are scattered by the specimen to form a 3-dimensional • cryoEM image. The sample is fixed and coated with a thin layer of a heavy metal such as platinum to form a replica of the specimen. This replica is then scanned with a thin beam of electrons and the quanitity of electrons scattered along each successive point of the specimen is measured by detectors which surround the sample (see Figure). Since the amount of electron scattering depends on the angle and depth of the surface relative to the beam, the image has highlights and shadows that give it a three dimensional appearance. The resolution of SEM is not very high (approximately 10 nm with an effective magnification of up to 20,000 times) and only suface features can be examined. Therefore, the technique is generally used to study whole cells or tissues. 14 A 3-dimensional appearance with higher resolutions than SEM can be obtained by TEM by shadowing. In this case the metal coating is applied at an angle resulting in a replica reflects the height and depth of the specimen. Shadowing is often used in conjunction with other techniques. For example, in freeze- fracture and freeze-etching cells are frozen in cryoprotectant and cut with a knife. Freeze-fracture will often split the lipid bilayer membranes which are then shadowed with platinum. Alternatively, in freeze- etching, the water is sublimated and replicas formed. Negative staining can be used to visualize macromolecules and Scanning Electron Microscope supramolecular structures such as virus particles or cytoskeletal filaments. The samples are placed on the electron transparent carbon grids and stained with heavy metals. Areas with biological structures appear more electron transparent. The fixation and manipulation of the specimen will often distort cells. Cryo-electron microscopy is used to overcome this problem. Special holders, which keep hydrated specimen at -160oC, allows viewing without fixation, staining or dehydration. 15 MICROSCOPY APPENDIX. ALDEHYDE FIXATIVES Formaldehyde and glutaraldehyde are a commonly used fixatives for both light and electron microscopy. Formaldehyde is a small molecule (HCHO). The formaldehyde monomers form polymers in aqueous solutions. The liquid known as formalin is 37-40% formaldehyde by weight and most of the polymers are 2-8 units long. Higher polymers (n up to 100) are insoluble in water and sold as a white powder called paraformaldehyde. To be useful as a fixative, the solution must contain monomeric formaldehyde. Dilution of formalin with a buffer at physiological pH results in an almost instantaneous formation of o monomers. Conversion of paraformaldehyde to monomers requires heat (typically 60 C) and the addition of hydroxide ions. Commercial formalin contains about 10% methanol and small amounts of formate ions, whereas a formaldehyde solution prepared from paraform- aldehye initially does contain any methanol or formate. Formaldehyde's mechanism of action is based on the reaction of the aldehyde group with primary amines in proteins. A cross-link between neighboring proteins can also be formed if the primary amines are close enough together. The initial reaction of formaldehyde with protein is complete within 24 hours, but the formation of cross-links, called methylene bridges, proceeds much more slowly (several weeks). Lipids, nucleic acids and carbohydrates are trapped in a matrix of insoluble and cross-linked proteins. In practical terms, formaldehyde penetrates tissues rapidly (because of its small size), but it slowly cross-links the proteins. Glutaraldehyde contains two aldehyde groups separated by three methylene bridges (HCO-CH -CHO). These two aldehyde groups and the flexible methylene bridge greatly 2 3 increases the cross-linking potential of glutaraldehyde over formaldehyde. In solution glutaraldehyde exists as polymers of various sizes which exhibit an enormous potential for cross-linking proteins (Figure). In contrast with formaldehyde, the chemical reaction of glutaraldehyde with protein is fast, but the penetration of tissue is slower, especially for the 16 larger oligomers. Therefore, an 'EM grade' glutaraldehyde, which contains low polymers, should be used for fixation. In addition, fixation with glutaraldehyde results many leftover free aldehyde groups which cannot be washed out of the tissue. For many applications these free aldehyde groups needs need to be removed or blocked. A common blocking method is to treat with glycine or another small primary amine. The combination of formaldehyde and glutaraldehyde is also used as a fixative for electron microscopy. This takes advantage of the rapid penetration of formaldehyde molecules, which quickly stabilizes the structure of the tissue, followed by a more thorough cross-linking of proteins mediated by the more slowly penetrating glutaraldehyde. 17 CHAPTER 2SPECTROPHOTOMETRY Spectrophotometry is a versatile analytical tool. The underlying principle of spectro- photometry is to shine light on a sample and to analyze how the sample affects the light. Advantages of spectrophotometry are: 1) it is often non-destructive (i.e., can measure and recover sample), 2) it is selective (often a particular compound in a mixture can be measured -14 without separation techniques), 3) it has a short time interval of measurement (10 seconds). SPECTROPHOTOMETRY THEORY Light can be described as a wave. This wave has an electric component and a magnetic component which are perpendicular to each other (Figure). Electromagnetic radiation exhibits a direction of propagation and wave-like pro- perties (i.e., oscillations). The energy of electromagnetic radiation is defined as: E = hc/λ = hυ where E = energy, h = Planck's constant, c = the speed of light, λ = the wave length, and υ = frequency. Light behaves both as a wave and as a particle. The conceptual particle of light is called a photon and is represented by hυ. Electromagnetic radiation exhibits a wide spectrum and specific ranges of wavelengths have names (Figure). The energy of electromagnetic radiation is inversely proportional to its wavelength. When a light wave encounters a particle, or molecule, it can be scattered (i.e., direction changed), absorbed (energy transferred), or unaffected. Molecules only absorb discreet packets of energy, or quanta, and absorption occurs when the energy of the photon corre- sponds to differences between energy levels in that particular mole- cule. These discrete energy levels, called electronic energy levels, are a property of the particular molecule and are determined by the spatial distribution of the electrons. Absorption of the energy from the photon elevates the molecule to an excited electronic state (see Figure in Chapter 3) by causing an electron to move from one orbit to another. These electronic energy levels are further subdivided into vibrational levels. The vibrational levels correspond to stretching and bending of various covalent bonds. The transitions to the excited 19 state can occur between different vibrational levels giving a range of energy that can be absorbed by the molecule. A molecule or substance that absorbs light is called a chromophore. Chromophores exhibit unique absorption spectra (Figure) and can be defined by a wavelength of maximum absorption, or λ , of a broad absorbtion band due to the vibrational levels. The absorption max spectra can consists of several absorption maxima of various amplitudes. A large number of biological molecules absorb light in the visible and ultraviolet (UV) range. The net affect of absorption is that the intensity of the light decreases as it passes through a solution containing a chromophore. The amount of light absorbed depends on the nature of the chromophore, the concentration of the chromophore, the thickness of the sample, and the conditions (eg., pH, solvent, etc.) under which absorption is measured. Absorption is governed by the Beer-Lambert Law: -εdc I = I10 or log(I/I) = -εdc o o where I = final light intensity, I = initial light intensity, ε = molar extinction coefficient, d = o thickness, and c = molar concentration. Absorption (A) will be defined by: A = -log(I/I) = εdc o The molar extinction coefficient (ε) is defined as the A of 1 M of pure compound under standard conditions and reflects something about the nature of the chromaphore. The units of ε are liter/cm⋅mole. However, the extinction coefficient can be expressed in other units. For example, it can be expressed in terms of mM concentration. The thickness of the sample (d) is almost always 1 cm and therefore can be ignored in calculations. Sometimes, though, the 2 extinction coefficient units is expressed in cm /mole (by converting liters to cubic centimeters) and care should be taken in making calculations. In cases where the molecular weight of the 1% 1% substance is not known, or varies, E is used as the extinction coefficient. E is defined as the A of a 1% (w/v) solution. It is important to precisely record the units of ε when looking it up or determining it experimentally since these units will determine the concentration. It is also important to record the conditions (eg., pH, solvent, temperature, etc.) for an extinction coefficient (see below). 20

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