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3 - Enzyme Kinetics, Proteomics, and Mass Spectrometry
- Bal Ram Singh, Raj Kumar
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Summary
Biochemical analysis techniques are a set of procedures and protocols used by scientists to analyze substances and the chemical reactions underlying life processes. For a complete biochemical analysis, the biomolecules such as proteins, lipids or carbohydrates must be isolated in their pure form from biological samples, characterized for structure–functions. Assays may involve spectrophotometric measurement, gel staining, densitometric analysis, determining the concentration and purity of the protein, and so on. The assays can be simple or tedious, based on the size, shape and net charge, in order to determine different properties and characteristics of the myriad biomolecules involved in different stages of life processes and to understand their role in the biochemical steps involved in maintaining the life.
Since biomolecules generally exist in very minute quantities in the cell, so, many a times it is not possible to employ chromatographic or other such methods to purify them in adequate quantities to carry out chemical and biological analyses. Hence, other approaches, such as proteomics have been devised so as to at least develop a general sense of the types of molecules, changes in their quantities (e.g. during growth), and identify their sequences using such sensitive techniques, such as mass spectroscopy, for comparison with known molecules, aiming at characterization of the biochemical processes in the cells by observing a few biomolecules at a time. This, in combination with the recent developments in understanding gene structures with their expressions, their genetic manipulations, bioinformatic tools, and large-scale automated analysis techniques, enable scientists to analyze cell process in greater depths.
One of the well-known biochemical processes that exists in every cell, and likely is responsible for the development, evolution, and maintenance of life is the enzymatic characteristics of most proteins, with some exception of RNA. Enzymes are used to assay biological activity of cells, tissues, organs, and even the whole organism or animal. Enzyme characteristics are useful tools for understanding structure–function relationship and biological activity at the molecular level. Therefore, a scientist interested in biochemistry generally has a solid knowledge of the principles and practice of enzyme function, including enzyme kinetics.
BASICS OF CHEMICAL KINETICS
Order and Molecularity
Chemical kinetics is a wonderful tool in understanding the behavior of a chemical reaction. To classify any chemical reaction, two parameters are very important: order and molecularity. Molecularity of any reaction is defined as a number of molecules that can alter the course of any reaction.
Appendix 2 - Laboratory Safety
- Bal Ram Singh, Raj Kumar
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Due to the wide variety of experiments listed in this text it would be cumbersome to roster each specific chemical danger here. Therefore, dangers inherent to each exercise are listed in the experiments in each chapter. The following safety precautions are a generalized list that are likely to be encountered in the exercises covered in the text. Individuals involved in the biochemical experimentation should familiarize themselves intimately with these precautions.
General Precautions
1. Follow the instructions and pay attention to all the steps from start to finish before beginning an experiment. Know the use of all the equipment in the lab before beginning the work.
2. The most important safety rule is to know the location of the safety equipment and how to use it. The equipment should be checked periodically to ensure that it is in working order. Remember the building evacuation protocol.
3. Wear safety goggles at all times in the laboratory. For biochemical experimentation regular eyeglasses do not provide sufficient protection from either chemical hazards or broken glassware. Recently, the American Chemical Society has approved the use of contact lenses but only when worn in combination with safety goggles. The contact/goggle combination is important because most modern eyewash stations are not able to remove chemicals trapped behind contacts.
4. Proper clothing should be worn in the laboratory at all times. Long sleeve shirts, long pants and full shoes are the best choice. Skin protection will be at a maximum if the aforementioned garments are covered with a full lab coat/apron.
5. Make use of the fume hoods for handling volatile and/or hazardous chemicals. When handling chemicals gloves should be worn to protect the hands.
6. Dispose of solid and liquid waste in containers, which are properly labeled. Notify the instructor if any of the waste containers are full or damaged. If you not sure where to dispose of the waste ask your instructor for help.
7. Get acquainted with the layout of the lab, paying special attention to the fire extinguishers, emergency eye wash stations, first aid kits and the nearest emergency phone. Knowledge of the location of this apparatus may help save your life and prevent injury to others.
8. Never eat or drink in the laboratory area. Contamination of food or drink can take place without your knowledge.
List of Tables
- Bal Ram Singh, Raj Kumar
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2 - Recombinant DNA and Protein Technology
- Bal Ram Singh, Raj Kumar
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Summary
Introduction
To understand cellular structures and their functions, comprehensive knowledge of proteins and the mechanisms of their action is needed. For better understanding of cellular processes, the protein component needs to be extracted from a complex mixture. Earlier, proteins were extracted in a purified form either by using large amounts of animal or (a variety of) plant tissues or biological fluids. With the advent of recombinant protein technology, most biochemical projects start with conceptualizing purification of the target protein in a recombinant form. The capabilities of this technology allows biochemists to design, clone, extract, and purify a protein for its biochemical characterization, better understanding, and research/commercial applications.
Protein purification is a series of steps required to isolate a target protein from a complex mixture of biomolecules. Protein purification may be preparative or analytical. Preparative purification aims to produce relatively large amount of protein for their use, such as soy protein extract and insulin. Analytical purification aims at obtaining just sufficient amounts of protein required for the specific research. In general, recombinant protein purification steps are straightforward. The process starts from identifying the gene of interest, which is a clone and transform into a proper host, and after induction of gene expression the designed protein is ready for further experimentation, such as development of purification protocols and its biochemical characterizations. However, these experiments involve several steps that can go wrong, such as inappropriate transformation, poor growth conditions, inclusion body formation, degradation of the target protein, inactive protein, and even no expression.
Inside tissue, some proteins are present in enough amounts that can be isolated from their host directly. But for getting larger amounts of proteins, scientists need to enhance their expression by using an appropriate host and develop protocols for their purification. There are four main steps involved: a gene of interest, an optimized vector that contains that gene, an appropriate expression host, and a complete purification procedure. Cloning, expression, and purification are the three major steps in getting the desired amount of pure protein.
Cloning
Gene cloning employs a series of molecular biology methods that culminate with the insertion of the gene of interest into a host cell and replicating this in many other cells under appropriate conditions of the expression of that gene.
1 - Introduction
- Bal Ram Singh, Raj Kumar
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Biotechnology – Its Background and History
The office of Technology Assessment of the U.S. Congress defines biotechnology as “any technique that uses living organisms or their products to make or modify a(another) product, to improve plants or animals, or to develop microorganisms for specific uses.” In its broadest terms, such a definition includes human beings, making biotechnology to be as old as the development of human skills such as to grow crops, harvest them, and use them as ingredients to cook their meals. In fact, a term Biotechnik, originally referring to social biology by Goldscheid in 1911, was used in the Nature journal of 1933 to print an editorial on Biotechnology (Bud, 1989; Goldcheid, 1911). Ironically, time has come a full circle as scientists are now examining gene expression patterns under different social conditions (Cole, 2014).
Biotechnology as a system of knowledge and application probably goes back to Vedic times, when the system of Ayurveda (literally meaning knowledge of life) was developed. Reference to the use of herbs for treating medical conditions are to be found in the earliest literature of India, over 14,000 years ago. According to BioREACH (which stands for Biotechnology Resource for Educational Advancement of Curriculum in High Schools at Arizona State University) some forms of biotechnology were being practiced by the ancients in Babylon, Egypt, and Rome in their selective breeding practices with livestock, over 10,000 years ago. There are instances where people around 6000 BC used fermentation to make wine and beer; and when the Chinese used lactic acid producing bacteria to produce yogurt around 4000 BC.
The modern term ‘biotechnology’ was first coined by a Hungarian engineer, Karl Ereky (1878–1952), in 1919. He defined biotechnology as general processes of converting raw materials into useful products, such as on industrial farms, using living organisms. A previous term ‘zymotechnology’ was used in the nineteenth century for using microorganisms to produce products like bread, wine, tofu, and so on. In the early twentieth century zymotechnology also included biological chemistry and covered usage of biological molecules such as enzymes, amino acids, and proteins for industrial production.
Modern biotechnology took root after the discovery of genetic material and the central theme of the gene progression route, namely DNA → RNA → Proteins, during the 1930s to the 1950s.
Color Plates
- Bal Ram Singh, Raj Kumar
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Index
- Bal Ram Singh, Raj Kumar
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Frontmatter
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5 - Molecular Biology
- Bal Ram Singh, Raj Kumar
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Introduction
Molecular biology is the study of the molecular basis of biological processes in living organisms. These include understanding the interactions between biomolecules that form the basis of life as well as the regulation of these interactions. Of particular significance to molecular biology are the nucleic acids (DNA/RNA), proteins and their synthesis. Nucleic acids are the fundamental constituents of a living cell where they function to encode and transmit genetic information needed for the continuity of life in every tissue and organism.
In just over 60 years since the discovery of the structure of DNA by James Watson and Francis Crick, and 40 years since venturing into the revolutionary area of genetic engineering, the concepts of molecular biology have gained a strong foothold in routine household conversations. DNA, RNA, 23andMe, and enzymes have become the subject of interesting discussions on television, newspapers, and business weeklies. The field of microbiology is important not just for scientists and practitioners in the field, but even common people seem curious for insights into the genomics, diagnostics and serology of pathogens, for example, after the outbreak of Covid-19 in 2019. The astonishing scientific discoveries in the field of molecular biology have led to an understanding of the complex biological processes of life and its evolution.
Heredity is the transmission of characteristics from one generation to the other in all forms of life on earth. Much of the understanding of heredity originated in the 1800s, from the pioneering research of Gregor Mendel, an Augustinian monk. He studied the characteristics of parents and offspring in pea plants and defined the “law of combination of different characters” which described the gene as a carrier of hereditary traits. The relation between genetic material and DNA, however, was not demonstrated until the middle of the twentieth century during which time scientists continued to explore the nature of genes, study the behavior of chromosomes during cell division, and the chemical composition of the nucleic acids. Several lines of evidence from a combination of studies carried out within a living organism (in vivo) and those performed in cell or tissues grown in culture or in cell extracts (in vitro), converged to define DNA as the unvarying bearer of heredity. This realization led to the elucidation of the structure of DNA, the molecule of life, and marked the beginning of the development of molecular biology.
Appendix 6 - Chapter Contributors
- Bal Ram Singh, Raj Kumar
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Chapter 1: Introduction … Raj Kumar and Bal Ram Singh
Chapter 2: Recombinant DNA and Protein Technology … Raj Kumar and Bal Ram Singh
Chapter 3: Enzyme Kinetics, Proteomics, and Mass Spectrometry … Ghuncha Ambrin, Raj Kumar and Bal Ram Singh
Chapter 4: Bioanalytical Techniques … Raj Kumar and Bal Ram Singh
Chapter 5: Molecular Biology … Roshan Vijay Kukreja, Raj Kumar and Bal Ram Singh
Chapter 6: Cell Culture … Ghuncha Ambrin, Raj Kumar and Bal Ram Singh
Chapter 7: Antibody Technology … Ghuncha Ambrin, Raj Kumar and Bal Ram Singh
List of Figures
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Appendix 1 - Troubleshooting: Cell Culture
- Bal Ram Singh, Raj Kumar
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Appendix 1.1 Problems and Solutions
The matter given below lists some potential problems and offers possible solutions that may help you troubleshoot your cell culture experiments. Note that the list given below includes only the most commonly encountered problems in cell culture and provides guidelines to solutions only.
Appendix 1.2 Equipment for Cell Culture
• Laboratory refrigerator/freezer combination. Separation of refrigerator from the freezer unit for tissue culture media and ingredients is recommended to minimize contamination.
• Two-stage vacuum trap for aspiration. Construct an aspirator for the culture hood using two Buchner or Erlenmeyer flasks, two 1-hole or 2-hole rubber stoppers, glass Pasteur pipettes or glass tubes, and Tygon® tubing. Use a 1000 ml or larger flask for the first stage trap. Connect the second stage to a vacuum supply or pump through a vacuum filter to protect the pump from residual water vapor.
• Stainless steel pipette sterilization boxes. Obtain at least two sterilization boxes and keep one autoclaved box of pipettes in the culture hood at all times (suggested model: Fisher scientific 03-475-5 rectangular box for 9 inch Pasteur pipettes).
• Set of pipettes for flow hood. Keep a separate set of autoclavable 2–20 μl, 20–200 μl, and 100–1000 μl pipettes to be used only for cell culture. Autoclave the pipettes according to the manufacturer's instructions. Autoclave repeatedly every one to two months or in case of contamination.
• Tissue culture microscope. An inverted, phase contrast microscope with 10x, 20x, and 40x phase-contrast objectives and a long working distance condenser should be used for observing the extent of cell extraction, for counting cells, and for cell maintenance.
• Clinical centrifuge. A fixed-angle or swinging bucket centrifuge for 15 ml conical tubes with a maximum speed of 1,300 g can be used for pelleting nematodes and floating eggs on sucrose (suggested model: Fisher scientific 228 benchtop centrifuge).
6 - Cell Culture
- Bal Ram Singh, Raj Kumar
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Introduction
Cell cultures are remarkable tools in biological and biochemical research. Cellular models are often used for understanding physiological processes, biochemical production, antibody production, vaccine production, and cancer research, along with a multitude of other uses. Additionally, they are also useful to understand the mechanism of signal transduction, protein synthesis, drug action (pharmacology and toxicology), drug metabolism, cell–cell interactions, and genetics. Furthermore, cell culture also provides future avenues for medical treatment including cell-based therapy and regenerative medicine. These are the reasons why in vitro propagation of cells becomes an essential requirement for every biochemical lab. Such technology provides a user-friendly and relatively cheap tool to examine the biological issues, overriding the legal, moral, and ethical concerns related to in vivo studies. In a cell culture experiment, cells are provided with a conducive environment to grow, different from their native environment. This is widely referred to as cell culturing and consists of several steps of isolating cells from their native environment; they will then be maintained under precise conditions and nurtured with additives, in an appropriate medium.
Cells are the smallest structural and functional units of any organism. After isolation from their respective tissues (plants and animals), they can be grown (cultured and differentiated) in an artificial media (mixture of buffers and nutrients). Ross Harrison (1907) first developed a technique for tissue culture called the “hanging drop technique.” He placed a small tissue in a medium (containing serum) from which cells migrated to the surrounding environment. Carrel and Lindbergh (1935), further developed cell culture technique. and then utilized it to build a vaccine. Their research revolutionized the study and improved understanding of cell cultures. In the 1940s and 1950s, several protocols for assays were developed to examine viral growth (Salvador Luria and Renato Dulbecco). Later, more techniques were developed to examine the different characteristics of cells including growth, differentiation, protein production, and cell death. Since its inception in the twentieth century, cell culture based techniques has been invaluable in the development of basic virology, vaccine development, disease modeling, pathogenesis and drug development.
Cells are isolated from animals, microbes or plants. Once isolated, all cells require aseptic techniques, and viable growth and proliferation conditions.
Appendix 4 - Significant Figures
- Bal Ram Singh, Raj Kumar
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In biochemistry, as in all other branches of chemistry, the reporting of data is, in many cases, as important as the experimentation, which took place to obtain the data. In our experience, in teaching the biochemistry laboratory course, we have found that many students feel that the strict adherence to reporting experimental data with the correct number of significant figures is a task only needed in the analytical chemistry laboratory. The lax attitude towards significant figures, scientific notation, accuracy and precision has caused much confusion for the students when reporting their experimental findings. Therefore, it is in the students’ best interest to review the following rules regarding the basic principles for reporting experimental findings:
Number of Significant Figures
In general, the number of significant figures pertains to the number of digits recorded for the value of a calculated or a measured sum. Ultimately, the number of significant figures is an indication of the precision of the measurement or calculation (precision will be discussed in statistics section).
If, for example, a student weighs the mass of a biological sample to be 0.998, 0.997, and 0.999 g for 3 separate measurements, a total of 3 significant figures would result from each of the measurements (zeros here are not significant). The fluctuation in the third decimal place indicates that this number is an estimate and is an indication of the balance limit. If a less accurate balance were used the measurements may look something like 1.0, 1.0, and 1.0 g. The measurements on the crude balance would result in each of the measurements having 2 significant figures (zeros here are significant). The aforementioned example illustrates the problem for most students, “which zeros are significant and which are not.” The rules for significant figures that follow should clarify any questions:
1. All digits are significant except zeros at the beginning of a number and possibly those at the end of a number (Rule 2 and 3 apply to terminal zeros).
2. All zeros, which terminate a number and are to the right of a decimal place are significant. For example, from our biological sample listed above, 1.0, 1.00, and 1.000 g. These examples contain 2, 3, and 4 significant figures respectfully.
Appendix 5 - Units in the Biochemistry Laboratory
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In the biochemistry laboratory course there is a strong emphasis on the manner in which data is reported to the instructor. As you may remember from your freshman chemistry laboratory experience, keeping a close watch on the units of your results will help you when calculations must be performed via dimensional analysis. The system of units most readily recognized and used by biochemists the world over is the International System of Units (SI). The SI system is rooted in 7 fundamental base units of measure (see Table A5.1). A description of the units, which are most regularly used in the biochemistry lab will follow. Biochemistry experiments and instrumentations mostly involve a range of different units. Students are required to know the multiple units and their prefix to report the quantities in conventional terms (Table A5.2).
Of the seven basic units listed in Table A5.1, the unit used most often in the biochemistry laboratory is the mole. The mole and its multiple unit forms mmol, μmol and nmol are put to use in biochemistry whenever the amount of some chemical is to be determined/reported. Closely associated with the use of the mole quantity, is mass and its accompanying units. Prior to the description of mass and the most used multiple unit forms of mass it is important for the biochemistry student to be reminded of the difference between mass and weight. In general, the mass of an object is a uniform measure of the quantity (amount) of matter in an object. Weight, on the other hand, is the attraction affinity between an object and its environment (object's placement on earth). Recall that weight (W) can be expressed in the following formula:
Inclusion of the gravity term in the above equation indicates that objects at higher altitudes will weigh less than the same object at low altitudes, while the mass of the object remains the same. In the biochemistry lab, the mass multiple unit form that a student will most likely encounter will be increasingly small amounts from grams (g) to nanograms (ng). You will most often encounter g when weighing solids for making mixtures. The smaller quantities of mg, μg and ng will be encountered when the mass of an isolated product is tabulated.
Contents
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7 - Antibody Technology
- Bal Ram Singh, Raj Kumar
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Introduction to Immunochemical Techniques
Life on the earth has an abundance of pathogenic and non-pathogenic microbes, which apart from mutual symbiotic and survival connections contain several toxic and allergenic molecules that bring about imbalance in homeostasis. These substances have a variety of mechanisms to disturb the functioning of their host. To counter these threats, mammals developed a complex and evolutionary matured array of immune and defensive mechanisms to check or annihilate these substances without damaging their own tissues. In general, there are two mechanisms which permit recognition and destruction of microbial, toxic, or allergenic substances: 1) Innate response: This is the first line of defense against the invading pathogen or toxin, and 2) Adaptive response: The second line of defense. Although the adaptive responses are temporary, they leave a memory associated with this process and for a specific antigen. The organism resorts to the same defense mechanism when it encounters the same antigen again. The adaptive response has the capability to regulate immune memory, and create an effective and specific host response against invading pathogens, even decades after the first encounter.
For identification of foreign material, immunoglobulins (IgG) and antibodies are integral parts of adaptive immune response in mammals. IgGs are present in the tissues and fluids of all vertebrates. Research related to antibodies started way back in 1890, when Emil von Behring and Shibasabura Kitasato began to immunize infected animals against diptheria. According to the side-chain theory, proposed by Paul Ehrlich in 1900, the pathogens bind to their side-chain receptors. Then, the modern era of antibody research and discovery started with examination at the atomic level of details of its structure (in 1973) and invention of monoclonal antibodies (in 1975).
The formation of an antigen–antibody complex, is due to a very specific interaction between antibody and its antigen, and is the basis of all immunochemical based technology. An antigen in general, is an exogenous substance that elicits an immune response and is recognized by very specific antibodies produced by the immunological responses to counter the invading antigen. They are usually either proteins or polysaccharides of high molecular weight. However, small molecules can also function as antigens, such as polypeptides, lipids, and nucleic acids. These small molecules (haptens) may generate immune response by coupling themselves to a larger “carrier protein”, such as bovine serum albumin or hemocyanin or other synthetic matrices.
4 - Bioanalytical Techniques
- Bal Ram Singh, Raj Kumar
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Introduction
Protein Science is the study of protein molecules by researchers from varied fields of science, including chemistry, physics, mathematics, and biology. The primary interest of such research is on the structure, function, design, and possible applications of protein molecules. Proteins are considered a part of family called biological macromolecules and before broaching their molecular structure, clarity about the definition of molecule is essential. Defining a molecule in biochemistry is a little different than in general chemistry. In general chemistry, a molecule normally comprises of two or more atoms bonded covalently in specific stoichiometry and defined geometry. For example, ethane, C2H6 has well defined stoichiometry and defined geometry. But this is not the case with the chiral molecule or cis-trans isomers of biochemistry.
In biochemistry, molecules are considered as components which may or may not be bonded together covalently in all parts. Local and non-local interactions play important roles in their structures. For example in a protein molecule there are several weak interactions taking place in the functional structure of that protein, apart from covalent bonds. Also in a complex protein, non-covalent interactions play roles in the very assembling of the constituent molecules. Take the case of apoptosomes and proteosomes. The geometry of biological molecules are unique 3-D arrangements of its components. There are various levels of structural organization of such biological macromolecules. These levels of complexity, in them, are described below:
Four degrees of structure
(a) Monomers are simple building blocks of macromolecules; and include sugars, amino acids and nucleotides.
(b) Primary structure (1°) is the linear rearrangement of residues in the covalently linked polymers.
(c) Secondary structure (2°) is a local regular structure, such as a-helix, b-sheets.
(d) Tertiary structure (3°) is a global 3-D fold or topology, such as native structure of protein.
(e) Quaternary structure (4°) is spatial arrangement of multiple distinct polymers, such as hemoglobin and proteosomes.
Biochemists devoted considerable effort over a very long time, employing a variety of biochemical techniques in order to understand the relationship between structure and function of biomolecules.
The current focus in life sciences is on learning more about proteomes with emphasis on the study of individual structures of biomolecules in order to understand their role in cellular functions. The tens of thousands of biomolecules encountered in living cells are mainly in two general groups.
Appendix 7 - Online Resources
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For the facilitation of students and readers relevant on-line resources have been listed below:
1. Protein structure prediction model including homology modeling, protein threading, and secondary structure prediction:
http://www.scfbioiitd.res.in/bhageerath/bhageerath_h.jsp
http://www.reading.ac.uk/bioinf/IntFOLD/
http://raptorx.uchicago.edu/
http://www.cbs.dtu.dk/services/CPHmodels/
https://arquivo.pt/wayback/20160514083149/http://toolkit.tuebingen.mpg.de/hhpred
https://toolkit.tuebingen.mpg.de/tools/hhpred
http://protein.ict.ac.cn/FALCON/
http://bioinf.cs.ucl.ac.uk/psipred/
https://www.predictprotein.org/
2. Protein yield and size:
https://www.expasy.org/resources/search/keywords:gel%20electrophoresis
3. Western blot:
https://www.licor.com/bio/image-studio-lite/
4. Antigen prediction tool:
https://www.genscript.com/antigen-design.html
http://tools.immuneepitope.org/bcell/
https://www.expasy.org/resources/search/keywords:epitope
5. Epitope analysis and mapping tool:
https://www.integralmolecular.com/epitope-mapping/?gclid=EAIaIQobChMIjcaZks-76wIVEovICh1NKwUHEAAYASAAEgIauvD_BwE
http://tools.iedb.org/main/
6. ELISA software and data analysis:
https://www.elisaanalysis.com/
https://www.mybiosource.com/my_assay_data_analyzing_software
7. 3D view inside the cell:
http://sciencenetlinks.com/tools/icell-app/
https://edshelf.com/tool/3d-cell-simulation-and-stain-tool/
8. Construction of cell membrane:
https://www.wisc-online.com/learn/natural-science/life-science/ap1101/construction-of-thecell-membrane
9. Analysis of mitotic defects, segmentation, tracking, feature extraction:
http://www.bioquant.uni-hd.de/bmcv/genomeresearch
http://www.csie.ntu.edu.tw/∼cjlin/libsvm
https://github.com/bnoi/MAARS/tree/master/doc/demos
10. Confluency measurement and evaluation:
https://opencirrus.org/
http://www.samba.org/ftp/rsync/rsync.html
https://doi.org/10.1371/journal.pone.0027672.g003
https://doi.org/10.1371/journal.pone.0027672.g004
https://doi.org/10.1371/journal.pone.0027672.t002
11. Enzyme kinetic analysis and rate analysis:
https://icekat.herokuapp.com/icekat
https://github.com/SmithLabMCW/icekat/blob/master/icekat/test.csv)
12. Designing enzymes:
https://www.creative-biolabs.com/computer-aided-enzyme-design.html
https://www.rosettacommons.org/software
Appendix 3 - Statistics
- Bal Ram Singh, Raj Kumar
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Summary
In the biochemistry laboratory, as in many other branches of chemistry, the statistical treatment of data is of the utmost importance for the accurate reporting of results. The unique nature of the biochemical sample places an even greater importance on said statistical processing. The biochemical sample is usually the result of many hours of chromatographic purification. The same sample usually degrades to an unusable status in a very short period of time and is more often than not at a very low concentration to begin. The biochemical sample characteristics listed above infer that characterization protocols must be performed quickly and repeated numerous times. Prior to the mention of the basic statistical analysis that will be used you will need to be familiar with the types of errors you will encounter in biochemical experimentation.
Statistical Terms and Definition
Accuracy: The closeness of an experimental measurement to the true value.
Precision: Is also sometimes called reproducibility, which is scattering of experimental value. The precision of a series of measurements refers to the closeness of the values obtained from the identical measurements of a quantity.
Errors in Biochemical Experimental Data
It would be impossible to explain the types of errors found in gathering biochemical data without asking the reader to keep in mind the definitions of Precision and Accuracy. With the above mentioned definitions at hand it is easier to delineate the following types of errors.
First, biochemical analysis can be affected by random (or indeterminate) errors. The effects of a random error can cause the results to be scattered around a mean value (the definition of mean will follow shortly). Ultimately, an indeterminate error is a reflection of the precision with which the data is measured. Most random errors arise in a system where the method of measurement is lengthened to its maximum sensitivity. The resulting random fluctuations in measurements are the effects of the random error contributions in total. In general, random errors of a measurement are the cumulative effects of many small random errors, which cannot be measured individually.
Second, systematic (or determinate) errors produce a mean value for a set of results that differs from an accepted value. Therefore, a determinate error causes the data from a series of measurements to vary either all high or low. Consequently, systematic errors are a reflection of the accuracy of the measurements.