Today most technicians, undergraduates and postgraduates in the biological and life sciences require knowledge of biochemistry and especially the practical aspects of the subject. Although, biochemistry constitutes one of the important subjects in the student's curriculum, techniques teaching have been somehow neglected for past many years in the institutions. One of the main reasons for such a poor status of this important subject is the paucity of trained teachers, particularly with biochemistry background. This has consequently hampered the practical curriculum in biochemistry at undergraduate as well as postgraduate levels. This book is aimed at undergraduates and postgraduates who have a basic grounding in biological sciences and are interested in a future career in research and industry. The book provides an understanding of up-to-date information on the concerned topics in a simple, lucid and concise manner. It attempts to convey something of the fascination of working in a field which overlaps the disciplines of biochemistry, cell-biology and biotechnology. The book content covers the various techniques used in biochemistry, molecular biology, microbiology, immunology, Pharmacology, laboratories. It deals with the comparative analysis, procedure followed for techniques, their advantages, drawbacks and limitations. The content covers about 90% of the various techniques used by Bachelors and Masters and Doctoral students moreover it is of use for students of various universities like: University of Delhi, Jamia Millia Islamia University, Banaras Hindu University, Bundelkhand University etc. The text of the book has been illustrated with simplified well-labeled color diagrams and pictures to make the subject easy to understand and interesting to the students. The book has potential for being developed as a textbook or Lab manual for techniques. The book is therefore suitable for many B.Sc., M.Sc and technical courses in biochemistry as per UGC model curriculum. It could also be used as source of reference for research workers who need to understand and use biochemical techniques in their work. The objective in writing this handbook is mainly to acquaint the beginner with various biochemistry principles and to provide a laboratory guide to teachers concerned. In writing the first edition, our aim is to explain a new and rapidly growing technology. The basic philosophy is to present the principles of gene manipulation and its associated techniques, in sufficient detail to enable the non-specialist reader to understand them. It is assumed that the reader would have a reasonable working knowledge of molecular biology
Additional Info
  • Publisher: Laxmi Publications
  • Language: English
  • ISBN : 978-93-83828-41-8
  • Chapter 1

    SAFETY IN LABORATORY Price 2.99  |  2.99 Rewards Points

    Laboratory safety may appear dull subject and the temptation may be to read it superficially or not at all. However, the view of subject changes rapidly if you find yourself in the middle of a fire or the victim of an accident and by this time ignorance can be dangerous or even fatal. Laboratories can be dangerous places in which to work and all users need to be aware of the potential hazards and to know what to do in cases of emergency. Before starting work in a new laboratory, it is important to get familiar with the layout of the room and the location of the safety equipment. The position of the emergency exits, fire alarm and extinguishers should be known so that appropriate action can be taken in the event of fire. The main taps for gas and water and the switch for electricity should also be located so that these services can be turned off in case of an emergency. Laboratory workers must also know the meaning of safety signs and symbols so they can be rapidly identified. They must also know the laboratory Do`s and Don'ts.
  • Chapter 2

    pH AND BUFFER SOLUTIONS Price 2.99  |  2.99 Rewards Points

    pH AND LIFE Many life forms thrive only in a relatively small pH range; an example of a buffer solution is blood. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. It is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. A mixture containing citric acid, potassium dihydrogen phosphate, boric acid, and diethyl barbituric acid can be made to cover the pH range 2.6 to 12.
  • Chapter 3

    PHYSICAL AND CHEMICAL TECHNIQUES Price 2.99  |  2.99 Rewards Points

    Techniques can be differentiated into separation and analytic. Separation techniques require two phases and the desired substance distributes itself between the two phases in a definite manner, and the separation is completed by physically separating the two phases. Various types include: Decantation, Filtration, Evaporation, Crystallization, Distillation, Fractional Distillation, Sublimation, Centrifugation, Chromatography, Electrophoresis. An analytical technique is a method that is used to determine the concentration of a chemical compound or chemical element. Various types include: 1. Titration, based on the quantity of reagent needed to react with the analyte. 2. Electroanalytical techniques, including potentiometry and voltammetry. 3. Based on the interaction of the matter with electromagnetic radiation—Spectroscopy: NMR, Optical methods (IR, UV)
  • Chapter 4

    CHROMATOGRAPHY Price 2.99  |  2.99 Rewards Points

    Chromatography Derived from Greek meaning ‘colored writing’. It is an analytical laboratory technique first used by Russian botanist Tswett to describe the separation of colored plant pigments on a column of alumina. It is always a biphasic system, where one phase is stationary while the other is mobile. Various types include paper chromatography, thin layer chromatography (TLC), column chromatography (CC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), liquid chromatography (LC), gas chromatography (GC), but all of these employ the same basic principle. In all types of chromatography, a sample of the mixture being analyzed, known as the analyte is applied and allowed to adhere to a stationary material known as the stationary phase, or adsorbent. A carrier fluid known as the mobile phase or eluent, is then made to flow through the adsorbent. Because the different components of the analyte exhibit varying degrees of strength of adhesion to the adsorbent, they also travel different distances through the adsorbent as the eluent flows through it, i.e., components adhering more strongly to the adsorbent travel more slowly than those with weaker adhesion. Thus resulting in separation of the various components of the analyte into individual samples that can be analyzed. Chromatography Derived from Greek meaning ‘colored writing’. It is an analytical laboratory technique first used by Russian botanist Tswett to describe the separation of colored plant pigments on a column of alumina. It is always a biphasic system, where one phase is stationary while the other is mobile. Various types include paper chromatography, thin layer chromatography (TLC), column chromatography (CC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), liquid chromatography (LC), gas chromatography (GC), but all of these employ the same basic principle. In all types of chromatography, a sample of the mixture being analyzed, known as the analyte is applied and allowed to adhere to a stationary material known as the stationary phase, or adsorbent. A carrier fluid known as the mobile phase or eluent, is then made to flow through the adsorbent. Because the different components of the analyte exhibit varying degrees of strength of adhesion to the adsorbent, they also travel different distances through the adsorbent as the eluent flows through it, i.e., components adhering more strongly to the adsorbent travel more slowly than those with weaker adhesion. Thus resulting in separation of the various components of the analyte into individual samples that can be analyzed.
  • Chapter 5

    MOLECULAR BIOLOGY Price 2.99  |  2.99 Rewards Points

    EXTRACTION OF DNA FROM BIOLOGICAL SAMPLES DNA isolation from biological tissue samples is the initial step for conducting pharmacogenetics studies. Since cells contain several components, methods adopted for DNA extraction should be able to separate the other components from DNA and at the same time ensure that the DNA is not denatured. There are several methods that can be used to separate DNA from biological samples and each method has its own advantage and disadvantage. Therefore, selecting a suitable method is dependent on factors like, the molecular weight of the target DNA, quantity and quality of DNA, time required, cost and whether the method requires the use of any hazardous chemicals etc. An important prerequisite before collecting the sample of blood/tissue for genotyping is that you have to take an informed written consent from the subject stating the purpose for sample collection. Confidentiality regarding sample storage is of utmost importance. For DNA extraction from blood samples following method is used in laboratory.
  • Chapter 6

    BLOTTING TECHNIQUES Price 2.99  |  2.99 Rewards Points

    SOUTHERN BLOTTING It is an analytical technique and the first blotting technique was developed by biologist Edwin Southern. It involves transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. First DNA is cut using Restriction Endonucleases into smaller fragments, which are then electrophoresed on agarose gel to separate according to size. Nylon or nitrocellulose membrane is placed on top of the gel and pressure is applied by placing wet stack of paper towels. Gel must not be allowed to dry. DNA is transferred to the membrane from the gel due to capillary action. The membrane is then baked or exposed to UV to permanently attach the transferred DNA to the membrane. The membrane is then exposed to a labeled (radioactive or enzyme) hybridization probe (a single stranded DNA fragment with a specific sequence whose presence in the target DNA is to be determined). After hybridization, excess probe is washed from the membrane and the pattern is visualized in case of a radioactive probe or by color development. Southern blotting technique is widely used to find specific nucleic acid sequence. Other blotting methods i.e., Western (proteins), Northern blot (RNA), Eastern blot (carbohydrate epitopes), Southwestern blot (DNA-binding proteins) that employ similar princi- ples, but using RNA or protein, have later been named in reference to Edwin Southern’s name.
  • Chapter 7

    MOLECULAR CLONING/RECOMBINANT DNA TECHNOLOGY/GENE MANIPULATION TECHNIQUES Price 2.99  |  2.99 Rewards Points

    CELL: ITS HEREDITARY COMPONENT Each cell in our body contains cytoplasm and the nucleus, the nucleus contains the hereditary material in the form of chromosomes. The two different nucleic acids present in the cell are ribonu- cleic acid (RNA) and deoxyribonucleic acid (DNA). A gene is a section of the chromosomal DNA which contains the information to make a specific polypeptide through the production of a specific RNA. DNA is a nucleic acid carrying the genetic instructions used in the development and func- tioning of all known living organisms and some viruses. The main function of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints or a recipe, or a code, since it contains the instructions needed to construct other cellular components, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but DNA also contains structural elements involved in regulating the use of this genetic information. DNA is a long polymer made from repeating units called nucleotides. The DNA chain is 22 to 26 Angstroms wide (2.2 to 2.6 nm) and one nucleotide unit is 3.3 angstrom (0.33nm) long. Although each individual repeating unit is very small, DNA polymers may carry millions of nucleotides.
  • Chapter 8

    SEQUENCING Price 2.99  |  2.99 Rewards Points

    Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases- adenine, guanine, cytosine, and thymine in a molecule of DNA. DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography.
  • Chapter 9

    IMMUNOCHEMICAL TECHNIQUES Price 2.99  |  2.99 Rewards Points

    THE IMMUNE RESPONSE Living organisms defend themselves against invading foreign substances by their immune system which recognizes and rejects the foreign cells and their products. The study of this immune response is the branch known as immunology which is further subdivided into cellular and humoral immunity. Cellular immunity: involves the ability of cells to recognize foreign substance and to respond by ingesting them so they are effectively removed from the organism. Humoral immunity: involves the complex proteins in the blood plasma which are able to react with and neutralize soluble foreign compounds of high molecular weight. Immunochemical techniques use this aspect of immune response.
  • Chapter 10

    SPECTROPHOTOMETRY Price 2.99  |  2.99 Rewards Points

    Colorimetry is the most widely used method for determining the concentration of compounds. This method makes use of the property that when a white light passes through a colored solution, some wavelengths are absorbed more than others. Many compounds are not themselves colored but can be made to absorb light in visible region by reaction with suitable reagents. The amount of light absorbed is proportional to the color intensity, which is proportional to the concentration of compound being measured. These are very specific and sensitive reactions and do not require complete isolation of compound and the constituents of a complex mixture like blood can be deter- mined after little treatment. This utilizes the Beer-Lambert’s law, i.e.,
  • Chapter 11

    FLUORESCENCE SPECTROSCOPY Price 2.99  |  2.99 Rewards Points

    George Gabriel Stokes named the phenomenon fluorescence in 1852. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. Energy is absorbed in the UV region of the spectrum and molecules are elevated from the ground state S0 to a high energy level S2. The excited molecules then return to the ground state with emission of visible light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. Aromatic compounds are capable of fluorescence, substituent ring is electron donating.
  • Chapter 12

    PHOTOSYNTHESIS AND RESPIRATION Price 2.99  |  2.99 Rewards Points

    Photosynthesis and Respiration: Living organisms require a continous supply of energy to maintain the varied functions. In most cases, this energy is obtained by the oxidation of metabolites from the digestion of food. 3 ways in which this oxidation can occur and they involve loss of electrons from the compound being oxidized. The chemical energy in food which is released during oxidation comes ultimately from light energy of sun captured during photosynthesis. In chloroplasts, light drives the conversion of water to oxygen and NADP+ to NADPH with transfer of H+ ions across chloroplast membranes. Photosynthesis is the process by which light energy is used to synthesize carbohydrate in plants and algae from CO2 and H2O. The synthesis of carbohydrate takes place in two stages called as light and dark reactions. Light reaction occurs only in presence of light which is absorbed by the green pigment chlorophyll present in chloroplasts. The light reaction consists of removal of electrons from water (photolysis) and these are then used to reduce NADP+ and generate ATP. There are 2 light driven reactions which takes place at reaction centers of photosystem I (PS I) and photosystem II (PS II) and operate in series. They consist of electron transport chains connected in zigzag Z- scheme. Dark reaction is the second stage of photosynthesis and doesnot require presence of light. This stage involves utilization of NADPH and ATP generated by light reaction to fix carbon dioxide
  • Chapter 13

    ENZYMES Price 2.99  |  2.99 Rewards Points

    Living organisms obtain and use energy very rapidly because of the presence of biological catalysts called enzymes. Like inorganic catalysts, enzymes change the rate of a chemical reaction but donot affect the final equilibrium; also small quantities are needed to carry out the reaction. But unlike inorganic catalysts, enzymes have a very narrow specificity, i.e. they can catalyse only a small range of reactions. Also, enzymes function under well-defined conditions of pH, temperature, substrate concentration, cofactors. Enzymes are named and classified according to the type of reaction catalysed. Main groups: Oxido-reductases, transferases, hydrolases, lyases, isomerases, ligases. In biochemistry, the Lineweaver–Burk plot (or double reciprocal plot) is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics, described by Hans Lineweaver and Dean Burk in 1934.
  • Chapter 14

    LIPIDS Price 2.99  |  2.99 Rewards Points

    Lipids are naturally occurring esters of long chain fatty acids, insoluble in water but soluble in acetone, alcohol, chloroform and ether. Saponification or alkaline hydrolysis gives alcohol and the sodium or potassium salts of the constituent fatty acids, these hydrolysis products may be water soluble. Classification into simple and compound lipids. Simple lipids or acylglycerols are esters of glycerol and fatty acids to give mono-, di- and triglycerides. Triglycerides are the predominant form in nature. A triglyceride (TG, triacylglycerol, TAG, or triacylglyceride) is an ester derived from glycerol and three fatty acids and type depends on the oil source vegetable oil (unsaturated) and animal fats (saturated). In humans, unused calories are stored as triglycerides and their concentration in blood increases with the consumption of high carbohydrate foods. In the intestine enzyme Pancreatic lipase acts and splits the triglycerides into monoacylglycerol and free fatty acids by lipolysis. TGs are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells and collected by the lymph sys- tem and transported to the large vessels near the heart before being mixed into the blood. Various tissues can capture the chylomicrons, releasing the TGs to be used as a source of energy. Fat and liver cells can synthesize and store triglycerides. When the body requires fatty acids as an energy source, the hormone glucagon signals the breakdown of the triglycerides by activating lipase to release free fatty acids. As the brain cannot utilize fatty acids as an energy source (unless converted to a ketone), the glycerol component of triglycerides can be converted into glucose, via glycolysis by conversion into Dihydroxyacetone phosphate and then into Glyceraldehyde 3-phos- phate, for brain fuel when it is broken down. So, if the brain‘s needs become high then, fat cells may also be broken down. TGs, as components of very-low-density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. In the human body, high levels of TGs in the bloodstream can cause atherosclerosis, the risk of heart disease and stroke. The risk can be due to inverse relationship between TG level and HDL-choles- terol level. Chylomicrons are lipoprotein particles that consist of triglycerides, phospholipids, cholesterol and proteins. They transport dietary lipids from the intestines to other locations in the body.
  • Chapter 15

    LIPIDS Price 2.99  |  2.99 Rewards Points

    Lipids are naturally occurring esters of long chain fatty acids, insoluble in water but soluble in acetone, alcohol, chloroform and ether. Saponification or alkaline hydrolysis gives alcohol and the sodium or potassium salts of the constituent fatty acids, these hydrolysis products may be water soluble. Classification into simple and compound lipids. Simple lipids or acylglycerols are esters of glycerol and fatty acids to give mono-, di- and triglycerides. Triglycerides are the predominant form in nature. A triglyceride (TG, triacylglycerol, TAG, or triacylglyceride) is an ester derived from glycerol and three fatty acids and type depends on the oil source vegetable oil (unsaturated) and animal fats (saturated). In humans, unused calories are stored as triglycerides and their concentration in blood increases with the consumption of high carbohydrate foods. In the intestine enzyme Pancreatic lipase acts and splits the triglycerides into monoacylglycerol and free fatty acids by lipolysis. TGs are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells and collected by the lymph sys- tem and transported to the large vessels near the heart before being mixed into the blood. Various tissues can capture the chylomicrons, releasing the TGs to be used as a source of energy. Fat and liver cells can synthesize and store triglycerides. When the body requires fatty acids as an energy source, the hormone glucagon signals the breakdown of the triglycerides by activating lipase to release free fatty acids. As the brain cannot utilize fatty acids as an energy source (unless converted to a ketone), the glycerol component of triglycerides can be converted into glucose, via glycolysis by conversion into Dihydroxyacetone phosphate and then into Glyceraldehyde 3-phos- phate, for brain fuel when it is broken down. So, if the brain‘s needs become high then, fat cells may also be broken down. TGs, as components of very-low-density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. In the human body, high levels of TGs in the bloodstream can cause atherosclerosis, the risk of heart disease and stroke. The risk can be due to inverse relationship between TG level and HDL-choles- terol level. Chylomicrons are lipoprotein particles that consist of triglycerides, phospholipids, cholesterol and proteins. They transport dietary lipids from the intestines to other locations in the body.
  • Chapter 16

    CARBOHYDRATES Price 2.99  |  2.99 Rewards Points

    A carbohydrate is an organic compound that consists only of carbon, hydrogen, and oxygen, usu- ally with a hydrogen : oxygen atom ratio of 2 : 1 (as in water) with the empirical formula Cm(H2O)n. (Exception deoxyribose, a component of DNA, has empirical formula C5H10O4.) Carbohydrates are not hydrates of carbon but it is more accurate to view them as polyhydroxy aldehydes and ketones. Carbohydrates/Saccharides are divided into four classes: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Monosaccharides and disaccharides are smaller (lower molecular weight) carbohydrates and are commonly referred to as sugars. Polysaccharides serve for the storage of energy (e.g., starch and glycogen), and as structural components (e.g., cellulose in plants and chitin in arthropods) and play key roles in the immune system, fertilization, prevent- ing pathogenesis, blood clotting, and development. The 5-carbon monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD, and NAD) and the backbone of the genetic molecule known as ribose in RNA and deoxyribose in DNA. Monosaccharides are the simplest carbohydrates and cannot be hydrolyzed to smaller carbohydrates. They are aldehydes or ketones with two or more hydroxyl groups. Monosaccharides are important fuel molecules as well as building blocks for nucleic acids, as n = 3, are dihydroxyacetone and D- and L-glyceraldehydes.
  • Chapter 17

    CELL MEMBRANES Price 2.99  |  2.99 Rewards Points

    The cell membrane or plasma membrane is a biological membrane separating the interior of all cells from the outside environment. Most of the lipid present in the membranes consists of phos- pholipid molecules associated together in a regular manner. This structural organization arises from the fact that phospholipids have hydrophobic and hydrophilic regions in the same molecule so also called amphipathic. Most phospholipids are also zwitterions since the phosphate group carries a negative charge at neutral pH and the base a positive charge (choline phosphoglyceride, ethanolamine phosphoglyceride) or a positive and negative charge (serine phosphoglyceride). The polar region of the phospholipids hydrophilic and seeks an aqueous environment, while the hydrophobic part of the molecule tends to increase its entropy by expelling water from its vicinity and associating together with hydrophobic regions of other molecules. Phospholipids thus orient themselves on the surface of an aqueous solution so that the polar region lies in water and the alkyl side chain in the air.

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