Term
| Cells obey the fundamental laws of ___________ |
|
Definition
|
|
Term
| The ______ ______ ______ ________ states that energy can neither be created nor destroyed. (conservation of energy. However, energy can be interconverted. An example is the internal combustion engine where chemical energy (bonds of the hydrocarbon molecules of the gasoline) is converted into mechanical energy (turning of the wheels). The vast majority of energy, however, is lost in the form of heat. Hydrocarbons are being oxidized by the gasoline. |
|
Definition
| First law of thermodynamics |
|
|
Term
| That portion of the universe which is being considered at the moment (cell, mitochondria, etc.) |
|
Definition
|
|
Term
| The rest of the universe that is not the portion of the universe which is being considered at the moment |
|
Definition
|
|
Term
| A system that is isolated from the environment. There is no exchange of matter and energy (chemistry) |
|
Definition
|
|
Term
| There is an exchange of matter and energy between the system and the environment (all biological systems) |
|
Definition
|
|
Term
| The transfer of energy from one place or form to another place or form by any process other than heat flow |
|
Definition
|
|
Term
| Amount of energy required to warm one gram of water one degree centigrade |
|
Definition
|
|
Term
| Concerned with the "direction" of a chemical reaction and enables one to predict whether or not a chemical reaction will occur spontaneously. Systems will spontaneously change from states of lower probability to states of higher probability. States of lower probability are more ordered, therefore, the universe changes and becomes more disordered. Temperature, free energy, enthalpy, and entropy are associated with this |
|
Definition
| The second law of thermodynamics |
|
|
Term
| The only quantity that can be measured on an absolute scale (second law of thermodynamics) |
|
Definition
|
|
Term
| The energy that drives a reaction and is considered to be the energy that is capable of performing work (second law of thermodynamics) |
|
Definition
|
|
Term
| The change in heat content (second law of thermodynamics) |
|
Definition
|
|
Term
| The degree of disorder (second law of thermodynamics) |
|
Definition
|
|
Term
| Spontaneous reactions will have a _________ free energy change - the higher the ______ value of ΔG, the greater the driving force of the reaction and the greater the amount of work that can be obtained from such a system |
|
Definition
|
|
Term
| In living systems there are no changes in temperature and pressure. Consequently, we define ________ _______ _______ as the gain or loss of calories when one moles of reactant is converted to one mole of product under standard conditions, which are one atmosphere of pressure and at 25°C |
|
Definition
| Standard free energy change (ΔG°) |
|
|
Term
| A spontaneous reactions (-ΔG°) |
|
Definition
|
|
Term
| A non-spontaneous reaction (ΔG°) |
|
Definition
|
|
Term
| All physical and chemical processes proceed in such a direction that the randomness or entropy of the universe increases to a maximum. Examples would be the eventual random diffusion of a drop of dye in an aquarium or the even distribution of heat in a bar exposed at one location to a flame |
|
Definition
| The second law of thermodynamics |
|
|
Term
| In any real process, ΔG will always be _________, while TΔG will be __________ |
|
Definition
|
|
Term
| May be defined as that fraction of the total energy change of a system that is available to do work as a system at constant temperature and pressure proceeds to the condition of equilibrium |
|
Definition
|
|
Term
| Just as the entropy of the system plus the surrounding proceeds to a ___________ as a process goes to equilibrium, the free energy of the system alone proceeds to a _________ |
|
Definition
|
|
Term
| Chemical reactions associated with biological organisms require the participation of specific molecules called __________ in order for the reaction to occur. The ________ enables the reactants to compensate for what is known as activation energy for the reaction |
|
Definition
|
|
Term
| The input of energy required to initiate a reaction that is an energy barrier that must be overcome in order for the reaction to occur. An enzyme will lower this sufficiently to allow a large percentage of the molecular population to react at only one point in time |
|
Definition
|
|
Term
| Required for biological/physiological interactions. Necessary for life. Cannot be produced by organisms. Some are water soluble and some are fat soluble |
|
Definition
|
|
Term
| Transfer of chemical group or electrons. An organic molecule that can serve as an intermediate carrier of chemical groups or electrons. The majority (8) are synthesized from vitamins. An example is Riboflavin (vitamin B2) which forms a part of NAD+, a critical component of the electron transport system of cellular respiration |
|
Definition
|
|
Term
| A non-protein structure (metal) that is required for the enzyme to function. An example is Na+ and the enzyme Na+ dependent ATPase. Control of different elements |
|
Definition
|
|
Term
| An intact and functional enzyme-cofactor complex |
|
Definition
|
|
Term
| The protein (enzyme) which remains after its cofactor has been removed. The enzyme is nonfunctional in the absence of its cofactor |
|
Definition
|
|
Term
| The number of substrate molecule transformed per unit time by a single enzyme molecule. Many enzymes have a turnover number of 1000 substrate molecules per second. There are some enzymes that can covert over 10,000 substrate molecule per second. Example: carbonic anhydras - 600,000; acetlycholinesterase - 25,000; DNA polymerase - 15. Determines how efficient the enzyme is at converting reactants to products |
|
Definition
|
|
Term
| Two or more enzymes that have as their primary function the catalysis of the same reaction (have same name). Are normally distributed in different regions of the body where one form is better suited for the conditions found in that particular organ. Performing exactly the same reaction occurring at different locations in the body |
|
Definition
|
|
Term
| The location on the enzyme where the substrate(s) binds. The three amino acids most frequently found here are serine, histidine, and aspartic acid |
|
Definition
|
|
Term
| All enzyme catalyzed reactions are characterized by an inability of higher and higher substrate concentrations to increase the reaction velocity beyond a definable or finite upper value |
|
Definition
|
|
Term
| Catalyzed reactions ________ become saturated at high substrate concentrations, while non-catalyzed reactions _________ |
|
Definition
|
|
Term
| The substrate concentration at which the velocity of the reaction is 1/2 maximum. Describes the affinity of an enzyme for its substrate. The smaller the value, the more avidly the enzyme will bind to the substrate in a dilute solution. The intracellular concentration of a substrate is approximately the same or greater than this value of the enzyme to which it binds. If this decreases, the affinity for substrate binding increases |
|
Definition
| Michaelis-Menton constant (Km) |
|
|
Term
| The reciprocal of the Michaelis-Menton equation |
|
Definition
|
|
Term
|
Definition
|
|
Term
| Removal of electrons, plus 1 or more protons |
|
Definition
|
|
Term
| Transfer of a phosphate from 1 substrate (usually ATP) to another. One of the most important enzymes in all of life |
|
Definition
|
|
Term
| Removal of a phosphate by hydrolysis |
|
Definition
|
|
Term
| Hydrolysis (breakdown of a molecule by the addition of water) A-B + H2O -> A-H + B-OH |
|
Definition
|
|
Term
| Removal of a carboxyl group, thus creating CO2, A-COOH -> AH + CO2 (Kreb's cycle produces CO2 in cellular respiration) |
|
Definition
|
|
Term
| There are many elements that can affect the ability of an enzyme to catalyze a reaction |
|
Definition
|
|
Term
| Involves the destruction or modification of one or more functional groups of the enzyme. There are a variety of ions or molecules that can function as being permanently changed |
|
Definition
|
|
Term
| Concerns a state of inhibition that can be reversed depending upon the conditions |
|
Definition
|
|
Term
| Inhibitor molecules compete with substrate molecules for binding to the active site of the enzyme. Not a permanent binding. How long it stays depends upon specific compound. Increase substrate concentration, decrease inhibitor concentration. Binding of the inhibitor does not necessarily shift the three dimensional structure of the enzyme. The inhibitor raises the Km value of the enzyme (less affinity between the substrate and enzyme). Examples are the organophosphates, such as malathion and parathion that have served as insecticides for decades |
|
Definition
| Competitive enzyme inhibition |
|
|
Term
| Inhibit the activity of acetylcholinesterase, a critical enzyme of the nervous system that degrades acetylcholine, an important neurotransmitter |
|
Definition
|
|
Term
| Inhibitor binds reversibly to a region of the enzyme other than the active site. The inhibitor may bind to the enzyme of the enzyme-substrate complex. Binding of the inhibitor changes the shape of the active site. The Km value for the enzyme is not affected by the inhibitor. The inhibitor decreases the Vmax value for the enzyme. Is not reversed by increasing the substrate concentration. Examples are reagents which combine reversible with -SH groups of cysteine residues that are essential for the catalytic activity of some enzymes (most are heavy metals, are individual ions: Cu++, Hg++, and Ag+) |
|
Definition
| Noncompetitive enzyme inhibition |
|
|
Term
| Some enzymes that require metal ions for activity may be ____________ by agents capable of binding to the essential metal |
|
Definition
|
|
Term
| The chelating agent ____________ ___________ reversibly binds Mg++ and other divalent cations and thus inhibits enzymes requiring such ions for activity |
|
Definition
| ethylendiamine tetracetate (EDTA) |
|
|
Term
| Inhibits some enzymes dependent upon Fe++ or Fe+++ by forming inactive complexes. Involved in electron transport of cellular respiration |
|
Definition
|
|
Term
| Many multienzyme systems possess the capacity for self-regulation of their overall reaction rate. In most such systems, the end product of the sequence of reactions can inhibit the first enzyme unique to the pathway. Thus, the rate of the entire sequence is determined by the steady-state concentration of the end product. |
|
Definition
|
|
Term
| Much larger and more complex than enzymes not possessing regulatory properties. All have more than one subunit (polypeptide chain). These enzymes have binding sites for the regulator molecule at a position other than the catalytic site. The end product (regulator) causes a change in the protein's shape, thus, altering its ability to bind to its substrate. Do not show typical inhibition kinetics (can not use Lineweaver-Burke plot) |
|
Definition
|
|
Term
| __________ chemical reactions require an input of energy and are thus linked to ____________ chemical reactions, which provide the energy to drive the reaction |
|
Definition
| Biosynthetic (anabolic), Biodegreadative (catabolic) |
|
|
Term
| In most instances, the required energy is stored as chemical bond energy in a small set of activated ______ ________, which contain one or more energy rich covalent bonds |
|
Definition
|
|
Term
| Store energy in an easily exchangeable form, either as a readily transferable chemical group or as high energy electrons, and they can serve a dual role as a source of both energy and chemical groups for biosynthetic reactions |
|
Definition
|
|
Term
| The most important of the activated carrier molecules are _____, _______, and ________ |
|
Definition
|
|
Term
| The widely used and versatile activated carrier molecule. It can both provide both energy (between -11 and 13 kcal/mole) and also as a provider of a phosphate(s) group during chemical reactions |
|
Definition
| Adenosine triphosphate (ATP) |
|
|
Term
| Reactions that involve the transfer of a phosphate group to a molecule is a __________ reaction. Are involved in many important cellular functions, including the activation of substrates and the regulation of cell signaling processes. |
|
Definition
|
|
Term
| Are important electron carriers during oxidation-reduction reactions and are commonly part of coupled or linked reactions in cells. Are each capable of picking up a "packet of energy" in the form of two high energy electrons plus a proton (H+), thus becoming ________ and ______, respectively. These molecules can therefore function as carriers of hydride ions (the H+ plus two electrons, or H-) |
|
Definition
| NAD+ (NADH), NADP+ (NADP) |
|
|
Term
| There are 20 naturally occurring ______ ______, which are the building blocks of proteins. All of these are found in nature, except for praline, have a free carboxyl group and a free un-substituted group attached to the alpha carbon. They each possess a characteristic "R" group. They crystallize from neutral aqueous solutions as dipolar ions, rather than as un-dissociated molecules. Optical isomers are possible, because of the alpha carbon |
|
Definition
|
|
Term
| Dipolar ions. Negative and positive components |
|
Definition
|
|
Term
| Alanine, valine, leucine isoleucine, phenylalanine, tryptophan, methionine, and proline. Hydrophobic and hydrocarbons |
|
Definition
|
|
Term
| Glycine, serine, threonine, cysteine, tyrosine, asparagines, and glutamine. Partial charges. |
|
Definition
| Polar, but uncharged amino acids |
|
|
Term
| Lysine, arginine, and histidine. Positive charge |
|
Definition
| Positively charged basic amino acids |
|
|
Term
| Aspartic acid and glutamic acid |
|
Definition
| Negatively charged acidic amino acids |
|
|
Term
| By far the most structurally complex and functionally sophisticated molecules known. Have four structural classifications. |
|
Definition
|
|
Term
| Indicates the number of polypeptide chains in a protein, the sequence of amino acids within them, and the location of inter and intra-chain disulfide bridges. When two or more polypeptide chains (subunits) are present in the molecule, they are usually held together by secondary forces. Disulfide bonds between cysteine side groups also function in holding the chains together |
|
Definition
| Primary protein structure |
|
|
Term
|
Definition
|
|
Term
| Connecting two proteins to one another |
|
Definition
|
|
Term
| The helical or extended structure of a polypeptide chain. Several different configurations of this, but the most common are the alpha helix and the beta pleated sheet. Alternating layers of alpha helix and beta pleated sheets |
|
Definition
| Secondary protein structure |
|
|
Term
| The most important arrangement of the polypeptide chain is the ______ _______, which is a helical pattern formed by the hydrogen bonding that occurs between groups on the same polypeptide chain. X-ray diffraction studies illustrate evidence for this pattern in a large variety of proteins |
|
Definition
|
|
Term
| Characterized by the presence of large numbers of glycine and alanine residues. Secondary forces such as hydrophobic interactions help to hold the structure in place. Silk is a good example |
|
Definition
|
|
Term
| Most important structural protein |
|
Definition
|
|
Term
| Basic chemistry of "R" groups determines whether you have a(n) ________ ______ or ________ _______ ______ |
|
Definition
| Alpha helix, beta pleated sheet |
|
|
Term
| Most important hydrophobic structural protein |
|
Definition
|
|
Term
| The three dimensional folding pattern of the polypeptide chain(s) that characterizes a protein in its native state. This configuration represents the energetically most favorable arrangement of the polypeptide chain. Each specific sequence of amino acids takes up the particular "native" arrangement that makes possible a maximum number of favorable atomic contacts between it and its environment. |
|
Definition
| Tertiary protein structure |
|
|
Term
| The presence of proline prevents the continuation of a helical pattern, thus it is interrupted. The formation of disulfide bridges between cysteine residues. When disulfide bridges are present, the helix undergoes a distortion. The most important reason for irregularity in protein structure arises from the diverse nature of the amino acid side groups. Each side group tends to form the energetically most favorable secondary interaction with other groups. |
|
Definition
| The majority of proteins do not exist in a purely helical configuration. Some have very few helical regions |
|
|
Term
| It is sensible to expect the ________ side groups to be found stacked next to each other in the interior of proteins, while the external surface contains side groups that are ________ |
|
Definition
|
|
Term
| That proteins assume a three dimensional configuration which represents the energetically most stable arrangement is evidenced by the fact that when proteins are subject to __________ ___________ (high temperature or urea and mercaptoethanol) the native state and functional capacity are lost |
|
Definition
|
|
Term
| A reducing agent used to break disulfide bridges |
|
Definition
|
|
Term
| All about waste product. End products are water and CO2. Important constituent of urine. Disrupts secondary reactions (ionic bonds, etc.) |
|
Definition
|
|
Term
| The sub-unit interactions and assembly for multimeric (at least two polypeptide chains) proteins. Proteins that demonstrate this are large molecule with molecular weights of at least 50,000 Daltons |
|
Definition
| Quaternary protein structure |
|
|
Term
| A compact and stable folded region of a polypeptide chain with certain combinations of the alpha helix and beta pleated sheet. These are distinct globular or fibrous regions, usually from 100 to 300 amino acid residues in length |
|
Definition
|
|
Term
| A particular combination of alpha helices and beta pleated sheets that has a characteristic sequence and is associated with a specific function |
|
Definition
|
|
Term
| Proteins are capable of a _________ of functions within biological organisms, primarily because of the vast number of different shapes and configurations they adopt |
|
Definition
|
|
Term
| Responsible for 1/3 of weight in bone in the human body. Prevents bones from becoming brittle |
|
Definition
|
|
Term
| Living cells contain thousands of different ________, each of which catalyzes one particular reaction. Examples include: tryptophan synthetase - makes the amino acid tryptophan; pepsin - degrades dietary proteins in the stomach; ribulose bisphosphate carboxylase - helps convert carbon dioxide into sugars in plants; DNA polymerase - copies DNA; protein kinase - adds a phosphate group to a protein molecule |
|
Definition
|
|
Term
| Outside cells, collagen and elastin are common constituents of extracelluar matrix and form fibers in tendons and ligaments. Inside cells, tubulin forms long, stiff microtubules and actin forms filaments that underlie and support the plasma membrane; alpha-keratin forms fibers that reinforce epithelial cells and is the major protein in hair and horn |
|
Definition
|
|
Term
| In the bloodstream, serum albumi carries lipids, hemoglobin carries oxygen , and transferrin carries iron. Many proteins embed in cell membranes transport ions or small molecules across the membrane |
|
Definition
|
|
Term
| Myosin in skeletal muscle cells provides the motive force for humans to move, kinesin interacts with microtubules to move organelles around the cell, dynein enables eucaryotic cilia and flagella to beat |
|
Definition
|
|
Term
| Iron is stored in the liver by binding to the small protein ferritin; ovalbumin in egg whit is used as a source of amino acids for the developing bird embryo; casein in milk is a source of amino acids for baby mammals |
|
Definition
|
|
Term
| Many of the hormones and growth factors that coordinate physiological functions in animals are proteins, insulin is a small protein that controls glucose levels in blood; netrin attracts growing nerve cells in specific direction in developing embryo |
|
Definition
|
|
Term
| Rhodopsin in the retina detects light, the acetylcholine receptor in the membrane of a muscle cell receives chemical signals released from a nerve ending; the insulin receptor allows liver cell to respond to the hormone insulin by taking up glucose |
|
Definition
|
|
Term
| The lactose repressor in bacteria silences the gene for the enzymes that degrade the sugar lactose |
|
Definition
|
|
Term
| Organisms make many proteins with highly specialized properties. These molecules illustrate the amazing range of functions that proteins can perform. The antifreeze proteins in Arctic and Antarctic fishes protect their blood against freezing |
|
Definition
|
|
Term
| Specific cellular organelles may be isolated and purified for investigation by means of |
|
Definition
| Cellular fractionation and centrifugation |
|
|
Term
| To disrupt tissues and cells by one of several mechanical procedures, including a method which shears the cells between a close fitting plunger and the walls of a glass vessel. This process, termed homogenization, is usually done in a sucrose solution |
|
Definition
|
|
Term
| Generates progressively high speeds and gravitational forces to fractionate the cell homogenate into their respective components |
|
Definition
| Differential centrifugation |
|
|
Term
| The pellet from a differential centrifugation spin is re-suspended in a buffer and then layered on top of a sucrose gradient (usually a 20-80 sucrose gradient). The solution is then centrifuged and the organelles of a particular type will migrate and eventually settle in a region of the sucrose gradient in which the density of the organelle is similar to that of the sucrose. Get organelle out of tube by puncturing side with a syringe |
|
Definition
| Density gradient centrifugation (equilibrium centrifugation) |
|
|
Term
| Techniques available for the separation and purification of various macromolecules will utilize specific physical or chemical properties of the molecule to obtain the separation. Usually the most common difference between species of macromolecules are _______ _______, _______ _______, _________, ______, and __________ |
|
Definition
| Molecular weight, ionic charge, solubility, shape, density |
|
|
Term
| The initial step in most separation experiments involves treatment of the homogenate or cell fraction with solutions that selectively dissolve or precipitate the desired material. For the extraction, add phenol, a detergent and a high concentration of salt to the solution that contains the DNA and shake well. The proteins will precipitate out of solution, while the DNA concentrates in the aqueous phase. Add ethanol to the aqueous phase. The ethanol will cause the DNA to precipitate (with RNA). Collect the DNA on a glass rod. Treat the sample with enzymes (proteases and RNAase) to remove unwanted proteins and RNA |
|
Definition
| Purification and separation of DNA |
|
|
Term
| Opens and separates up the phospholipid membrane (and any other membranes) |
|
Definition
|
|
Term
| Proteins will precipitate out in the presence of this |
|
Definition
|
|
Term
| Separation of water and this. Will remain in aqueous state |
|
Definition
|
|
Term
| Focuses on the fact that particles in suspension (cells, organelles, or molecules) having different masses or densities will settle toward the bottom of this type of tube at different rates. The behavior of a particle in this type of field depends chiefly upon its weight and the resistance it encounters in moving through the suspension medium. Thus size, density, and shape influent the movement in most types of this. In order for particles to sediment from solution, the force must overcome the forces of diffusion, which maintain the particles evenly dispersed in the solution. |
|
Definition
|
|
Term
| The weight of a sample (expressed in grams) |
|
Definition
|
|
Term
| A ratio of the sample's weight to its volume (expressed in grams per liter) |
|
Definition
|
|
Term
| ____________ _______ is proportional to the _____________ ___________ of the rotor (measured in revolutions per minute - rpm) and the distance of the centrifuge tube from the center of the rotor |
|
Definition
| Centrifugal force, rotational rate |
|
|
Term
| Proteins vary greatly in their _______, but not in their _________ |
|
Definition
|
|
Term
| The ultracentrifuge was developed by ________ ________ in 1925 |
|
Definition
|
|
Term
| This instrument operates under a vacuum and the chamber containing the rotor is refrigerated. The instrument is capable of extremely high centrifugal forces, ie, 250,000 times the force of gravity |
|
Definition
|
|
Term
| When the centrifugal force on molecules in solution greatly exceeds the opposing force of diffusion, the molecules will sediment downward toward the bottom of the centrifuge tube. The molecule, such as a protein, will migrate at a constant rate, which is characterized as the ___________ __________ and it is a function of both the weight and shape of the molecule |
|
Definition
| Sedimentation coefficient (s) |
|
|
Term
| Proteins have sedimentation coefficient values in the range of _____ to _______ seconds. |
|
Definition
|
|
Term
| A sedimentation coefficient value of 1*10^-13 seconds is called a |
|
Definition
|
|
Term
| Two of the more commonly employed types of centrifugation by cell biologist are |
|
Definition
| Density gradient centrifugation and velocity sedimentation centrifugation |
|
|
Term
| Uses a preformed gradient, usually 20-80% of a solution, such as sucrose. A solution containing the sub-cellular components that are to be separated are carefully layered on top of the gradient (the 20% portion). During centrifugation, the particles (organelles) sediment down the density gradient at their own rate that is determined primarily by their weight. Each group of specific particles (organelles) will settle in a region of the sucrose in which their densities are similar. Each group of the particles (organelles) will form bands or zones in the sucrose. Can be used to determine the molecular weight of a protein. |
|
Definition
| Density gradient centrifugation |
|
|
Term
| Normally used for separating molecules and/or organelles, but it can not be used to determine sedimentation coefficients or the molecular weight of a protein. The separations are on the basis of differences in buoyant density of the particles (organelles). The separation is independent of the size or shape of the particles. A preformed gradient is not used. Instead, a high concentration of a salt is employed, such as a 6M solution of cesium chloride (CsCl). At equilibrium (hours later) a gradient of CsCl will be formed in the tube. The particles (organelles) will have separated according to their density into bands. Each component will reach equilibrium at a position where its density exactly equals that of the CsCl solution. Cesium chloride has a high solubility and a low viscosity. The bands can be located optically or by analyzing samples as they pass from the bottom of the tube |
|
Definition
| Velocity sedimentation centrifugation |
|
|
Term
| Historically, one of the most widely used and effective techniques used by cell biologists to separate and purify proteins has been ___________ |
|
Definition
|
|
Term
| A solution that contains the protein flows by gravity through a column that has been packed with highly hydrated polmeric carbohydrate based beads. Globular proteins will be sorted out because proteins of different sizes will differ in their ability to penetrate pores that are distributed all over the particle's surface. The pores are of a uniform diameter |
|
Definition
| Gel-filtration (molecular exclusion) chromatography |
|
|
Term
| Highly hydrated polmeric carbohydrate based beads. Purchased according to their "G" value which is associated with the molecular weight of proteins. A G75 particle possesses pores of such a diameter that all proteins of a molecular weight equal to or greater than 75,000 Daltons will not be able to penetrate the pores as the molecules migrate down the column. The proteins that penetrate the pores will flow more slowly than the proteins that are too large to penetrate the pores. Consequently, proteins of a molecular weight greater than 75,000 Daltons will flow from the column before those proteins with molecular weight less than 75,000 Daltons |
|
Definition
|
|
Term
| The fluid that exits the bottom of the column may be analyzed for the presence of protein by measuring the concentration of the protein in successive small fractions by using light absorption measurements taken at _________ (using a spectrophotometer) |
|
Definition
|
|
Term
| Allows for the separation of proteins into two major groups on the basis of the molecular weight of the molecules |
|
Definition
| Gel-filtration chromatography |
|
|
Term
| The column is packed with small beads that carry either all positive or all negative charges that repel proteins of the opposite charge. As the solution migrates down the column, proteins of a charge different from that on the surface of the bead will be attracted to the bead and ionically bind to it |
|
Definition
| Ion exchange chromatography |
|
|
Term
| The proteins are dissociated from the beads by means of a _______ _________ or by ________ ________ ________ (high ionic strength). The effluent is collected and the presence of proteins is determined by the spectrophotometer |
|
Definition
| pH change, concentrated salt solutions |
|
|
Term
| This is the most specific method of isolating and purifying proteins. The column is packed with beads that have covalently attached to their surface either a specific substrate or antibody. As the solution flows through the column, the objective is to bind to the surface of the bead either the enzyme of the substrate or antigen of the antibody. Either may be removed by a pH change or by concentrated salt solutions |
|
Definition
|
|
Term
| A widely used technique to separate and isolate proteins in a solution. In essence, a heterogeneous solution of proteins is placed on a matrix, usually a polyacrylamide gel (PAGE) and exposed to an electrical field, with a positive electrode on one side and a negative electrode on the other. The proteins will migrate towards the anode at a speed that reflects both the size and net charge of the molecule. Larger proteins will migrate slower than smaller proteins |
|
Definition
|
|
Term
| The proteins may be separated in their ________ ________ or in a ________ ________. If the separation will occur with the proteins are in their _______ _______, the solution of proteins is carefully placed onto the PAGE, a current is applied to the gel through a buffer solution and the separation occurs over a period of hours |
|
Definition
| Native state, denatured state |
|
|
Term
| If the separation will take place with the proteins in a denatured state, a _______ such as _______ ______ _______ may be used to disrupt weak secondary forces maintaining the shape of the protein, while mercaptoethanol may be used to reduce the disulfide bridges and completely denature the proteins |
|
Definition
| detergent, sodium dodecyl sulfate (SDS) |
|
|
Term
| Individual polypeptide chains form a complex with negatively charged molecules of SDS. Consequently, a negatively charged SDS-protein complex migrates through the gel. The proteins migrate at a rate that reflects their _________ _________ |
|
Definition
|
|
Term
| Plasma minus the blood clotting factors |
|
Definition
|
|
Term
|
Definition
|
|
Term
| Used to determine whether a specific (unique) protein is present. A complex technique that uses the resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. If a physician is attempting to determine whether a patient is infected with HIV, he would draw a sample of blood and then place a small amount of the blood serum onto a sodium dodecyl sulfate - polyacrylamide gel (SDS-PAGE) and run an electrophoresis profile to start the technique |
|
Definition
| Western blot (immunoblotting) |
|
|
Term
| Conduct electrophoreses (SDS-PAGE) to separate the proteins. After the run is complete, place the gel in a container that has a buffer present. Lay a nitroscellulose membrane onto the gel. The proteins will transfer to the nitrocellulose membrane |
|
Definition
| Electrotransfer of Western blot |
|
|
Term
| Incubate the membrane in a solution that contains an antibody (rabbit, anti-HIV which is the primary antibody) that has been raised in a rabbit against the protein to be detected (HIV antigen). If an HIV protein is present (person is infected) then the antibody will bind to the HIV protein |
|
Definition
| Primary antibody detection of western blot |
|
|
Term
| After rinsing the membrane, incubate it in a solution that contains an antibody (goat anti-rabbit which is the secondary antibody) that has been raised in a goat against a rabbit antibody. If the primary antibody is present (person is infected) then the secondary antibody will bind to the primary antibody. The secondary antibody has attached to it a specific enzyme (frequently acid phosphatase is used) which will be used next |
|
Definition
| Secondary antibody detection of western blot |
|
|
Term
| A solution that contains the substrate of the enzyme (acid phosphatase) is then exposed to the membrane. If the secondary antibody/enzyme complex is present, then a reaction will take place and a product will be produced. The product which will be in the form of a participate, will be a particular color (brown if the enzyme is acid phosphatase). If a color is not detected then the protein of interest (HIV protein) is not present and the patient is not infected with HIV |
|
Definition
| Enzyme detection of western blot |
|
|
