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M.Pharma (Quality Assurance)
Bio EvaluationBio EvaluationBio EvaluationBio Evaluation
Biochemical analysis of Drugs
Submiteed by
M.Pharma (Quality Assurance)
DATEDATEDATEDATE
Bio EvaluationBio EvaluationBio EvaluationBio Evaluation TechniquesTechniquesTechniquesTechniques
AssignmentAssignmentAssignmentAssignment onononon
Biochemical analysis of Drugs
Submitted to- Me
(Asst.Prof ccp landr
Submiteed by-Shmmon Ahmad & Jassjeet Kharha
(M.Pharm Q.A)
DATEDATEDATEDATE----14/03/201314/03/201314/03/201314/03/2013
TechniquesTechniquesTechniquesTechniques
Biochemical analysis of Drugs
Metreyi Sharma
(Asst.Prof ccp landran)
Shmmon Ahmad & Jassjeet Kharha
(M.Pharm Q.A)
M.Pharma (Quality Assurance)
Biochemical analysis techniques of drugs
“Biochemical analysis techniques refer to a set of methods, assays, and
procedures that enable(give a power) scientists to analyze the
substances found in living organisms and the chemical reactions
underlying life processes.”
To perform biochemical analysis of a biomolecule in a biological
system, there is a need to design a strategy to detect the biomolecule &
isolate it in pure form.
BIOMOLECULES:
A biomolecule is any molecule that is produced by a living organism,
including large macromolecules such as proteins, polysaccharides,
lipids, and nucleic acids, as well as small molecules such as primary
metabolites and natural products.
Types of biomolecules
• Lipids,protein, polysaccharides, glycolipids,
sterols, glycerolipids
• Vitamins
• Hormones, neurotransmitters
• Metabolites
• Monomers, oligomers and polymers
Most biomolecules occur in minute amounts in the cell, and for their
detection and analysis it is required to purify them from any
contamination. The methods for purification of biomolecules includes
simple precipitation, centrifugation, and gel electrophoresis,
chromatographic and affinity techniques.
M.Pharma (Quality Assurance)
BIOCHEMICAL ANALYSIS TECHNIQUES
• Spectrophotometery
• Chromatography
• Electrophoresis
• Radioimmuno assay
• Hyberidoma
• ELISA
• Centrifugation
Technician performing biochemical analysis
typically needs to design a strategy to detect that biomolecule, isolate it in pure
form. from among thousands of molecules that can be found in an extracts from a
biological sample, characterize it, and analyze its function. An assay, the
biochemical test that characterizes a molecule, whether quantitative or semi-
quantitative, is important to determine the presence and quantity of a biomolecule
at each step of the study. Detection assays may range from the simple type of
assays provided by spectrophotometric measurements and gel staining to
determine the concentration and purity of proteins and nucleic acids, to long and
tedious bioassays that may take days to perform.
The description and characterization of the molecular components of the cell
succeeded in successive stages, each one related to the introduction of new
technical tools adapted to the particular properties of the studied molecules. The
first studied biomolecules were the small building blocks of larger and more
complex macromolecules, the amino acids of proteins, the bases of nucleic acids
and sugar monomers of complex carbohydrates.
The molecular characterization of these elementary components was carried out
thanks to techniques used in organic chemistry and developed as early as the
nineteenth century. Analysis and characterization of complex macromolecules
proved more difficult, and the fundamental techniques in protein and nucleic acid
and protein purification and sequencing were only established in the last four
decades.
Most biomolecules occur in minute amounts in the cell, and their detection and
analysis require the biochemist to first assume the major task of purifying them
from any contamination. Purification procedures published in the specialist
literature are almost as diverse as the diversity of biomolecules and are usually
written in sufficient details that they can be reproduced in different laboratory with
similar results. These procedures and protocols, which are reminiscent of recipes in
cookbooks have had major influence on the progress of biomedical sciences and
were very highly rated in scientific literature.
M.Pharma (Quality Assurance)
The methods available for purification of biomolecules range from simple
precipitation, centrifugation, and gel electrophoresis to sophisticated
chromatographic and affinity techniques that are constantly undergoing
development and improvement. These diverse but interrelated methods are based
on such properties as size and shape, net charge and bioproperties of the
biomolecules studied.
Centrifugation procedures impose, through rapid spinning high centrifugal forces
on biomolecules in solution, and cause their separations based on differences in
weight. and refer to the process where biomolecules are separated because they
adopt Electrophoresis techniques take advantage of both the size and charge of
biomolecules different rates of migration toward positively (anode) or negatively
(cathode) charged poles of an electric field. Gel electrophoresis methods are
important steps in many separation and analysis techniques in the studies of DNA,
proteins and lipids. Both western blotting techniques for the assay of proteins and
southern and northern analysis of DNA rely on gel electrophoresis. The completion
of DNA sequencing at the different human genome centers is also dependent on
gel electrophoresis. A powerful modification of gel electrophoresis called
twodimensional gel electrophoresis is predicted to play a very important role in the
accomplishment of the proteome projects that have started in many laboratories.
Chromatography techniques are sensitive and effective in separating and
concentrating minute components of a mixture and are widely used for quantitative
and qualitative analysis in medicine, industrial processes, and other fields. The
method consists of allowing a liquid or gaseous solution of the test mixture to flow
through a tube or column packed with a finely divided solid material that may be
coated with an active chemical group or an adsorbent liquid.
The different components of the mixture separate because they travel through the
tube at different rates, depending on the interactions with the porous stationary
material.
Various chromatographic separation strategies could be designed by modifying the
chemical components and shape of the solid adsorbent material. Some
chromatographic columns used in gel chromatography are packed with porous
stationary material, such that the small molecules flowing through the column
diffuse into the matrix and will be delayed, whereas larger molecules flow through
the column more quickly.
Along with ultracentrifugation and gel electrophoresis, this is one of the methods
used to determine the molecular weight of biomolecules. If the stationary material
is charged, the chromatography column will allow separation of biomolecules
M.Pharma (Quality Assurance)
according to their charge, a process known as ion exchange chromatography. This
process provides the highest resolution in the purification of native biomolecules
and is valuable when both the purity and the activity of a molecule are of
importance, as is the case in the preparation of all enzymes used in molecular
biology.
The biological activity of biomolecules has itself been exploited to design a powerful
separation method known as affinity chromatography. Most biomolecules of interest
bind specifically and tightly to natural biological partners called ligands: enzymes
bind substrates
and cofactors, hormones bind receptors, and specific immunoglobulinscalled
antibodies can be made by the immune system that would in principle interact with
any possible chemical component large enough to have a specific conformation. The
solid material in an affinity chromatography column is coated with the ligand and
only the biomolecule that specifically interact with this ligand will be retained while
the rest of a mixture is washed away by excess solvent running through the column.
Once a pure biomolecule is obtained, it may be employed for a specific purpose such
as an enzymatic reaction, used as a therapeutic agent, or in an industrial process.
However, it is normal in a research laboratory that the biomolecule isolated is novel,
isolated for the first time and, therefore, warrants full characterization in terms of
structure and function. This is the most difficult part in a biochemical analysis of a
novel biomolecule or a biochemical process, usually takes years to accomplish, and
involves the collaboration of many research laboratories from different parts of the
world.
Recent progress in biochemical analysis techniques has been dependant upon
contributions from both chemistry and biology, especially molecular genetics and
molecular biology, as well as engineering and information technology. Tagging of
proteins and nucleic acids with chemicals, especially fluorescent dyes, has been
crucial in helping to accomplish the sequencing of the human genome and other
organisms, as well as the analysis of proteins by chromatography and mass
spectrometry.
Biochemical research is undergoing a change in paradigm from analysis of the role of
one or a few molecules at a time, to an approach aiming at the characterization and
functional studies of many or even all biomolecules constituting a cell and eventually
organs. One of the major challenges of the post-genome era is to assign functions to
all of the gene products discovered through the genome and cDNA sequencing
efforts.
The need for functional analysis of proteins has become especially eminent, and this
has led to the renovated interest and major technical improvements in some protein
separation and analysis techniques. Two-dimensional gel electrophoresis, high
M.Pharma (Quality Assurance)
performance liquid and capillary chromatography as well as mass spectrometry are
proving very effective in separation and analysis of abundant change in highly
expressed proteins. The newly developed hardware and software, and the use of
automated systems that allow analysis of a huge number of samples simultaneously, is
making it possible to analyze a large number of proteins in a shorter time and with
higher accuracy. These approaches are making it possible to study global protein
expression in cells and tissues, and will allow comparison of protein products from
cells under varying conditions like differentiation and activation by various stimuli
such as stress, hormones, or drugs.
A more specific assay to analyze protein function in vivo is to use expression systems
designed to detect protein-protein and DNA-protein interactions such as the yeast and
bacterial hybrid systems. Ligand-receptor interactions are also being studied by novel
techniques using biosensors that are much faster than the conventional
immunochemical and colorimetric analyzes.
The combination of large scale and automated analysis techniques, bioinformatic
tools, and the power of genetic manipulations will enable scientists to eventually
analyze processes of cell function to all depths.
BIOCHEMICAL ANALYSIS OF DRUGS:
Biochemical analysis of phospholipase D.
Phospholipase D (PLD) is distributed widely in nature, being present in various
isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the
hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic
acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation
reaction in which water is replaced by a primary alcohol to yield a phosphatidyl
alcohol. This reaction is exclusive to PLD and is employed as a specific assay for
the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the
release of choline from PC can be utilized. This chapter describes production of a
recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect
cells and its purification. It also provides details of the assay procedure in the
presence and absence of regulatory proteins in vitro. The assay of the enzyme in
cells in vivo is also documented using labeling of endogenous PC by incubating
the cells with (3)H-labeled fatty acid. Details of the assay utilizing the
transphosphatidylation reaction are presented. In this, 1-butanol is employed as the
primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer
chromatography of lipid extracts from the cells. A variation of this assay is
described using deuterated 1-butanol (1-butanol-d(10)) and detection of the
synthesized deuterated phosphatidylbutanol species by mass spectrometry.
Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity
M.Pharma (Quality Assurance)
based on fluorescence are also described. Many of the materials for these assays
are available commercially, with the exception of the fluorescently labeled DAG
substrate, which can be synthesized enzymatically in a simple one-step procedure
Biochemical analysis of proteins
In the field of protein and peptide biochemistry study, BioCentrum offers broad
spectrum of electrophoretic analysis (SDS-PAGE, native electrophoresis, IEF,
2DE, WB) and chromatographic analysis (FPLC, HPLC). Moreover, we offer
ability to determine the amino-acid composition of proteins and peptides, N-
terminal sequence determination by Edman degradation, determination of
secondary structure of proteins by circular dychroism (CD) and tertiary structure of
proteins by X-Ray crystallography. Additionally, we offer analysis of protein-
protein interaction by ELISA test and analysis of protein samples by MS.
Electrophoretic pattern
Liquid chromatography services
Amino acid analysis
Sequencing of proteins and peptides
Analysis of CD spectra
ELISA tests
Amino acid analysis
Our laboratory offers amino acid analysis of proteins and peptides for determination of prote
tryptophan nor cysteine is determined, no physiological nor modified amino acids are analyz
gas-phase using 6M HCl during 24 h at 115 Celsius centigrades. Released amino acids are co
(PTC) derivatives and analysed on PicoTag 3.9x150 mm column (Waters) installed on a Wa
is on a level of about 10 picomols but we suggest sending of a minimum 100 picomols of pro
proteins or peptides immobilized on a PVDF membranes. The samples for amino acid analys
advices presented at sequencing of liquid samples section.
The protein samples were hydrolyzed in gas phase using 6M HCl at 115 deg. C for
24 h. The liberated amino acids were converted into phenylthiocarbamyl (PTC)
derivatives and analyzed by high-pressure liquid chromatography (HPLC) on a
PicoTag 3.9x150 mm column (Waters, Milford, MA, USA)."
M.Pharma (Quality Assurance)
ELISA test
ELISA is one of the most commonly used biochemical assay, applicable both in
research and diagnostics. BioCentrum offers range of services in the field of ELISA
optimization, including optimization of assay conditions such as blocking solution,
time of incubation with antibodies and serum antibodies. Analyses are performed
using fluorimetric and spectrophotometric detection.
Biochemical Analysis use UV/Visible spectroscopy for Bioresearch laboratories
routinely use UV/Visible spectroscopy for the analysis of proteins, enzymes,
nucleic acids and oligonucleotides. Whether a quick check of optical density,
DNA-purity, enzyme analysis, or a quantitative determination of a protein or DNA,
the Lambda series of UV/V is spectrometers can quickly and easily perform your
analysis.
These reliable workhorse systems are controled by our powerful but easy-to-use
UV WinLab™ Software. Proven in thousands of installations around the world,
UV WinLab can scan a spectrum, collect wavelength programed data, work in
concentration mode or collect time-drive data. It has a sophisticated report
generator that takes advantage of stored report templates. The system can be
operated from user created methods or from the bio specific methods supplied.
A large number of ready-to-run methods for routine biochemical analysis are
included with our UV BioLab™ collection. With a single mouse-click the method
is activated and the instrument is ready to measure your samples. Simply call-up
the method and start it. The UV BioLab collection of pre-programmed methods is
available at no extra charge for all Lambda systems, and include five key method
groups:
• Nucleic acid analysis
• Protein analysis
• Kinetic analysis
• General quantitative analysis
• General UV/Vis spectroscopic analysis
Quick and Easy Protein Analysis
Protein analysis and most common colorimetric protein assays can be done quickly
and easily with the pre-programmed methods included with our UV BioLab
M.Pharma (Quality Assurance)
collection. Simply prepare the standard solutions and samples according to
protocol, and activate the respective method. The measurements will automatically
be taken with one simple click of the mouse. Oligocalculator results for the
concentration and Tm of a 25mer oligonucleotide together with its Molecular Mass
and Molar Extinction Coefficient.
The result page of DNA purity (Ratio A260/A280) and concentration
determination.The following protein methods are included with the UV BioLab
collection. If sample automation is desired, a sipper system may be added. In
addition to the provided method, developers may choose to create their own
methods with UV WinLab software.
• OD280 for direct protein determination
• Lowry protein method for high and low concentration range
• Dye binding protein assay according to Bradford (see Figure 4, Coomassie blue)
• Biuret method for protein Quantification
• BCA assay
• Warburg/Christian method for
direct protein determination at 280/260 nm. Automated Kinetic Experiments With
UV WinLab’s integrated UV KinLab™ module and pre-programmed methods, it
is easy to monitor enzyme reactions in order to determine enzyme activity.
Absorbance versus time is displayed on-line, and enzyme activity calculated from
the resulting slope of the reaction curve. This can be done automatically with a
defined time interval, or calculated post-run with experiment specific timing
Automation of kinetic measurements in UV/Vis spectrometry is usually achieved
by use of manual or automated cell changers. Enzyme tests are often time-
consuming with a typical test lasting between 3 and 15 minutes. Automated
thermostatted Cell Changers can help reduce the measurement time for multiple
samples.
Cell Changer Systems for Any ApplicationOur 8+1 and 9+1 Cell Changer systems
significantly increase sample throughput and are optimized for time-dependent
UV/Vis spectroscopic measurements like enzyme kinetics, they may however be
used for all basic methods:
• Timedrive and Kinetics for single wavelength measurement.
M.Pharma (Quality Assurance)
• Wavelength program for measurement at up to 8 different wavelengths
including result calculation.
BIOCHEMICAL ANALYSIS OF HUMAN DNA2
ATPase assay
Purified hDna2 was incubated in 20 µl of reaction buffer containing 40 mM Tris–
HCl, pH 7.5, 5 mM MgCl2, 25 mM NaCl, 2.5 mM DTT, 0.1 mg/ml BSA, 5%
glycerol (v/v), 1 µg of oligonucleotide (primer, 22 bases) and various
concentrations of [γ-32
P]ATP at 37°C. These conditions were determined as
optimal in separate titrations of ATP, NaCl and time. The reaction was stopped by
adding EDTA to a final concentration of 4 mM, and the reaction mix (0.5 µl) was
spotted onto a polyethyleneimine cellulose plate (SELECTO SCIENTIFIC), which
was then developed in 0.5 M LiCl, 1 M formic acid solution. The results were
analyzed using the STORM PhosphoImager.
Nuclease assay
In general, nuclease activities of hDna2 were measured using a standard reaction
mixture (20 µl) containing 50 mM Tris–HCl, pH 7.5, 25 mM NaCl, 2 mM DTT,
0.25 mg/ml BSA, the 5′- or 3′-32
P-labeled DNA substrate, and various
concentrations of MgCl2 and ATP as indicated in the figure legends. NaCl
inhibited the nuclease activity (20 times inhibition at 125 mM) but 25 mM NaCl
was minimally inhibitory and included in the reactions to stabilize the
oligonucleotide substrates. After incubation at 37°C for 15 min, reactions were
stopped with 2× denaturing termination dye (95% deionized formamide, 10 mM
EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol), and boiled for 5 min.
The cleavage products were separated on a 12% sequencing gel (SequaGel,
National
Diagnostics) using Model S2 electrophoresis apparatus (BRL, 39 cm plate) and
analyzed using the PhosphoImager. Products were quantified using the
ImageQuant software on the phosphorimager, Substrate cleaved (%) is calculated
as follows: Substrate cleaved (%) = (product bands)/(substrate bands + product
bands) × 100.
Helicase assay
Helicase assays were performed with the nuclease-deficient mutant of hDna2
(D294A). The standard reaction mixtures contained 50 mM Tris–HCl, pH 7.5, 25
mM NaCl, 2 mM DTT, 0.25 mg/ml BSA, 4 mM MgCl2, 4 mM ATP and 32
P-
labeled helicase substrate. After incubation at 37°C for 1 h, reactions were stopped
with 5× stop solution (60 mM EDTA, 40% sucrose, 0.6% SDS, 0.25%
bromophenol blue and 0.25% xylene cyanole FF). Reaction products were then
M.Pharma (Quality Assurance)
separated using 8% native polyacrylamide gels containing 0.1% SDS, and detected
with PhosphoImager.
hDna2 nuclease substrates
All oligonucleotides were synthesized by Integrated DNA Technologies
(Coralville, IA). Oligonucleotide sequences are listed in Table 1. The locations of
biotinylation are indicated as underlined nucleotides in the table. The 5′ and 3′ end
labeling of oligonucleotides were performed as described previously (33).
Oligonucleotides were annealed as described in the figure legends to form various
structures. Flap substrate for the 5′–3′ nuclease assay were made by annealing a
downstream oligonucleotide, template, and upstream oligonucleotide at molar ratio
of 1:2:4. The upstream oligonucleotide was omitted to make the forked substrate.

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Biochemical analysis techniques

  • 1. M.Pharma (Quality Assurance) Bio EvaluationBio EvaluationBio EvaluationBio Evaluation Biochemical analysis of Drugs Submiteed by M.Pharma (Quality Assurance) DATEDATEDATEDATE Bio EvaluationBio EvaluationBio EvaluationBio Evaluation TechniquesTechniquesTechniquesTechniques AssignmentAssignmentAssignmentAssignment onononon Biochemical analysis of Drugs Submitted to- Me (Asst.Prof ccp landr Submiteed by-Shmmon Ahmad & Jassjeet Kharha (M.Pharm Q.A) DATEDATEDATEDATE----14/03/201314/03/201314/03/201314/03/2013 TechniquesTechniquesTechniquesTechniques Biochemical analysis of Drugs Metreyi Sharma (Asst.Prof ccp landran) Shmmon Ahmad & Jassjeet Kharha (M.Pharm Q.A)
  • 2. M.Pharma (Quality Assurance) Biochemical analysis techniques of drugs “Biochemical analysis techniques refer to a set of methods, assays, and procedures that enable(give a power) scientists to analyze the substances found in living organisms and the chemical reactions underlying life processes.” To perform biochemical analysis of a biomolecule in a biological system, there is a need to design a strategy to detect the biomolecule & isolate it in pure form. BIOMOLECULES: A biomolecule is any molecule that is produced by a living organism, including large macromolecules such as proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites and natural products. Types of biomolecules • Lipids,protein, polysaccharides, glycolipids, sterols, glycerolipids • Vitamins • Hormones, neurotransmitters • Metabolites • Monomers, oligomers and polymers Most biomolecules occur in minute amounts in the cell, and for their detection and analysis it is required to purify them from any contamination. The methods for purification of biomolecules includes simple precipitation, centrifugation, and gel electrophoresis, chromatographic and affinity techniques.
  • 3. M.Pharma (Quality Assurance) BIOCHEMICAL ANALYSIS TECHNIQUES • Spectrophotometery • Chromatography • Electrophoresis • Radioimmuno assay • Hyberidoma • ELISA • Centrifugation Technician performing biochemical analysis typically needs to design a strategy to detect that biomolecule, isolate it in pure form. from among thousands of molecules that can be found in an extracts from a biological sample, characterize it, and analyze its function. An assay, the biochemical test that characterizes a molecule, whether quantitative or semi- quantitative, is important to determine the presence and quantity of a biomolecule at each step of the study. Detection assays may range from the simple type of assays provided by spectrophotometric measurements and gel staining to determine the concentration and purity of proteins and nucleic acids, to long and tedious bioassays that may take days to perform. The description and characterization of the molecular components of the cell succeeded in successive stages, each one related to the introduction of new technical tools adapted to the particular properties of the studied molecules. The first studied biomolecules were the small building blocks of larger and more complex macromolecules, the amino acids of proteins, the bases of nucleic acids and sugar monomers of complex carbohydrates. The molecular characterization of these elementary components was carried out thanks to techniques used in organic chemistry and developed as early as the nineteenth century. Analysis and characterization of complex macromolecules proved more difficult, and the fundamental techniques in protein and nucleic acid and protein purification and sequencing were only established in the last four decades. Most biomolecules occur in minute amounts in the cell, and their detection and analysis require the biochemist to first assume the major task of purifying them from any contamination. Purification procedures published in the specialist literature are almost as diverse as the diversity of biomolecules and are usually written in sufficient details that they can be reproduced in different laboratory with similar results. These procedures and protocols, which are reminiscent of recipes in cookbooks have had major influence on the progress of biomedical sciences and were very highly rated in scientific literature.
  • 4. M.Pharma (Quality Assurance) The methods available for purification of biomolecules range from simple precipitation, centrifugation, and gel electrophoresis to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. These diverse but interrelated methods are based on such properties as size and shape, net charge and bioproperties of the biomolecules studied. Centrifugation procedures impose, through rapid spinning high centrifugal forces on biomolecules in solution, and cause their separations based on differences in weight. and refer to the process where biomolecules are separated because they adopt Electrophoresis techniques take advantage of both the size and charge of biomolecules different rates of migration toward positively (anode) or negatively (cathode) charged poles of an electric field. Gel electrophoresis methods are important steps in many separation and analysis techniques in the studies of DNA, proteins and lipids. Both western blotting techniques for the assay of proteins and southern and northern analysis of DNA rely on gel electrophoresis. The completion of DNA sequencing at the different human genome centers is also dependent on gel electrophoresis. A powerful modification of gel electrophoresis called twodimensional gel electrophoresis is predicted to play a very important role in the accomplishment of the proteome projects that have started in many laboratories. Chromatography techniques are sensitive and effective in separating and concentrating minute components of a mixture and are widely used for quantitative and qualitative analysis in medicine, industrial processes, and other fields. The method consists of allowing a liquid or gaseous solution of the test mixture to flow through a tube or column packed with a finely divided solid material that may be coated with an active chemical group or an adsorbent liquid. The different components of the mixture separate because they travel through the tube at different rates, depending on the interactions with the porous stationary material. Various chromatographic separation strategies could be designed by modifying the chemical components and shape of the solid adsorbent material. Some chromatographic columns used in gel chromatography are packed with porous stationary material, such that the small molecules flowing through the column diffuse into the matrix and will be delayed, whereas larger molecules flow through the column more quickly. Along with ultracentrifugation and gel electrophoresis, this is one of the methods used to determine the molecular weight of biomolecules. If the stationary material is charged, the chromatography column will allow separation of biomolecules
  • 5. M.Pharma (Quality Assurance) according to their charge, a process known as ion exchange chromatography. This process provides the highest resolution in the purification of native biomolecules and is valuable when both the purity and the activity of a molecule are of importance, as is the case in the preparation of all enzymes used in molecular biology. The biological activity of biomolecules has itself been exploited to design a powerful separation method known as affinity chromatography. Most biomolecules of interest bind specifically and tightly to natural biological partners called ligands: enzymes bind substrates and cofactors, hormones bind receptors, and specific immunoglobulinscalled antibodies can be made by the immune system that would in principle interact with any possible chemical component large enough to have a specific conformation. The solid material in an affinity chromatography column is coated with the ligand and only the biomolecule that specifically interact with this ligand will be retained while the rest of a mixture is washed away by excess solvent running through the column. Once a pure biomolecule is obtained, it may be employed for a specific purpose such as an enzymatic reaction, used as a therapeutic agent, or in an industrial process. However, it is normal in a research laboratory that the biomolecule isolated is novel, isolated for the first time and, therefore, warrants full characterization in terms of structure and function. This is the most difficult part in a biochemical analysis of a novel biomolecule or a biochemical process, usually takes years to accomplish, and involves the collaboration of many research laboratories from different parts of the world. Recent progress in biochemical analysis techniques has been dependant upon contributions from both chemistry and biology, especially molecular genetics and molecular biology, as well as engineering and information technology. Tagging of proteins and nucleic acids with chemicals, especially fluorescent dyes, has been crucial in helping to accomplish the sequencing of the human genome and other organisms, as well as the analysis of proteins by chromatography and mass spectrometry. Biochemical research is undergoing a change in paradigm from analysis of the role of one or a few molecules at a time, to an approach aiming at the characterization and functional studies of many or even all biomolecules constituting a cell and eventually organs. One of the major challenges of the post-genome era is to assign functions to all of the gene products discovered through the genome and cDNA sequencing efforts. The need for functional analysis of proteins has become especially eminent, and this has led to the renovated interest and major technical improvements in some protein separation and analysis techniques. Two-dimensional gel electrophoresis, high
  • 6. M.Pharma (Quality Assurance) performance liquid and capillary chromatography as well as mass spectrometry are proving very effective in separation and analysis of abundant change in highly expressed proteins. The newly developed hardware and software, and the use of automated systems that allow analysis of a huge number of samples simultaneously, is making it possible to analyze a large number of proteins in a shorter time and with higher accuracy. These approaches are making it possible to study global protein expression in cells and tissues, and will allow comparison of protein products from cells under varying conditions like differentiation and activation by various stimuli such as stress, hormones, or drugs. A more specific assay to analyze protein function in vivo is to use expression systems designed to detect protein-protein and DNA-protein interactions such as the yeast and bacterial hybrid systems. Ligand-receptor interactions are also being studied by novel techniques using biosensors that are much faster than the conventional immunochemical and colorimetric analyzes. The combination of large scale and automated analysis techniques, bioinformatic tools, and the power of genetic manipulations will enable scientists to eventually analyze processes of cell function to all depths. BIOCHEMICAL ANALYSIS OF DRUGS: Biochemical analysis of phospholipase D. Phospholipase D (PLD) is distributed widely in nature, being present in various isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation reaction in which water is replaced by a primary alcohol to yield a phosphatidyl alcohol. This reaction is exclusive to PLD and is employed as a specific assay for the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the release of choline from PC can be utilized. This chapter describes production of a recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect cells and its purification. It also provides details of the assay procedure in the presence and absence of regulatory proteins in vitro. The assay of the enzyme in cells in vivo is also documented using labeling of endogenous PC by incubating the cells with (3)H-labeled fatty acid. Details of the assay utilizing the transphosphatidylation reaction are presented. In this, 1-butanol is employed as the primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer chromatography of lipid extracts from the cells. A variation of this assay is described using deuterated 1-butanol (1-butanol-d(10)) and detection of the synthesized deuterated phosphatidylbutanol species by mass spectrometry. Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity
  • 7. M.Pharma (Quality Assurance) based on fluorescence are also described. Many of the materials for these assays are available commercially, with the exception of the fluorescently labeled DAG substrate, which can be synthesized enzymatically in a simple one-step procedure Biochemical analysis of proteins In the field of protein and peptide biochemistry study, BioCentrum offers broad spectrum of electrophoretic analysis (SDS-PAGE, native electrophoresis, IEF, 2DE, WB) and chromatographic analysis (FPLC, HPLC). Moreover, we offer ability to determine the amino-acid composition of proteins and peptides, N- terminal sequence determination by Edman degradation, determination of secondary structure of proteins by circular dychroism (CD) and tertiary structure of proteins by X-Ray crystallography. Additionally, we offer analysis of protein- protein interaction by ELISA test and analysis of protein samples by MS. Electrophoretic pattern Liquid chromatography services Amino acid analysis Sequencing of proteins and peptides Analysis of CD spectra ELISA tests Amino acid analysis Our laboratory offers amino acid analysis of proteins and peptides for determination of prote tryptophan nor cysteine is determined, no physiological nor modified amino acids are analyz gas-phase using 6M HCl during 24 h at 115 Celsius centigrades. Released amino acids are co (PTC) derivatives and analysed on PicoTag 3.9x150 mm column (Waters) installed on a Wa is on a level of about 10 picomols but we suggest sending of a minimum 100 picomols of pro proteins or peptides immobilized on a PVDF membranes. The samples for amino acid analys advices presented at sequencing of liquid samples section. The protein samples were hydrolyzed in gas phase using 6M HCl at 115 deg. C for 24 h. The liberated amino acids were converted into phenylthiocarbamyl (PTC) derivatives and analyzed by high-pressure liquid chromatography (HPLC) on a PicoTag 3.9x150 mm column (Waters, Milford, MA, USA)."
  • 8. M.Pharma (Quality Assurance) ELISA test ELISA is one of the most commonly used biochemical assay, applicable both in research and diagnostics. BioCentrum offers range of services in the field of ELISA optimization, including optimization of assay conditions such as blocking solution, time of incubation with antibodies and serum antibodies. Analyses are performed using fluorimetric and spectrophotometric detection. Biochemical Analysis use UV/Visible spectroscopy for Bioresearch laboratories routinely use UV/Visible spectroscopy for the analysis of proteins, enzymes, nucleic acids and oligonucleotides. Whether a quick check of optical density, DNA-purity, enzyme analysis, or a quantitative determination of a protein or DNA, the Lambda series of UV/V is spectrometers can quickly and easily perform your analysis. These reliable workhorse systems are controled by our powerful but easy-to-use UV WinLab™ Software. Proven in thousands of installations around the world, UV WinLab can scan a spectrum, collect wavelength programed data, work in concentration mode or collect time-drive data. It has a sophisticated report generator that takes advantage of stored report templates. The system can be operated from user created methods or from the bio specific methods supplied. A large number of ready-to-run methods for routine biochemical analysis are included with our UV BioLab™ collection. With a single mouse-click the method is activated and the instrument is ready to measure your samples. Simply call-up the method and start it. The UV BioLab collection of pre-programmed methods is available at no extra charge for all Lambda systems, and include five key method groups: • Nucleic acid analysis • Protein analysis • Kinetic analysis • General quantitative analysis • General UV/Vis spectroscopic analysis Quick and Easy Protein Analysis Protein analysis and most common colorimetric protein assays can be done quickly and easily with the pre-programmed methods included with our UV BioLab
  • 9. M.Pharma (Quality Assurance) collection. Simply prepare the standard solutions and samples according to protocol, and activate the respective method. The measurements will automatically be taken with one simple click of the mouse. Oligocalculator results for the concentration and Tm of a 25mer oligonucleotide together with its Molecular Mass and Molar Extinction Coefficient. The result page of DNA purity (Ratio A260/A280) and concentration determination.The following protein methods are included with the UV BioLab collection. If sample automation is desired, a sipper system may be added. In addition to the provided method, developers may choose to create their own methods with UV WinLab software. • OD280 for direct protein determination • Lowry protein method for high and low concentration range • Dye binding protein assay according to Bradford (see Figure 4, Coomassie blue) • Biuret method for protein Quantification • BCA assay • Warburg/Christian method for direct protein determination at 280/260 nm. Automated Kinetic Experiments With UV WinLab’s integrated UV KinLab™ module and pre-programmed methods, it is easy to monitor enzyme reactions in order to determine enzyme activity. Absorbance versus time is displayed on-line, and enzyme activity calculated from the resulting slope of the reaction curve. This can be done automatically with a defined time interval, or calculated post-run with experiment specific timing Automation of kinetic measurements in UV/Vis spectrometry is usually achieved by use of manual or automated cell changers. Enzyme tests are often time- consuming with a typical test lasting between 3 and 15 minutes. Automated thermostatted Cell Changers can help reduce the measurement time for multiple samples. Cell Changer Systems for Any ApplicationOur 8+1 and 9+1 Cell Changer systems significantly increase sample throughput and are optimized for time-dependent UV/Vis spectroscopic measurements like enzyme kinetics, they may however be used for all basic methods: • Timedrive and Kinetics for single wavelength measurement.
  • 10. M.Pharma (Quality Assurance) • Wavelength program for measurement at up to 8 different wavelengths including result calculation. BIOCHEMICAL ANALYSIS OF HUMAN DNA2 ATPase assay Purified hDna2 was incubated in 20 µl of reaction buffer containing 40 mM Tris– HCl, pH 7.5, 5 mM MgCl2, 25 mM NaCl, 2.5 mM DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), 1 µg of oligonucleotide (primer, 22 bases) and various concentrations of [γ-32 P]ATP at 37°C. These conditions were determined as optimal in separate titrations of ATP, NaCl and time. The reaction was stopped by adding EDTA to a final concentration of 4 mM, and the reaction mix (0.5 µl) was spotted onto a polyethyleneimine cellulose plate (SELECTO SCIENTIFIC), which was then developed in 0.5 M LiCl, 1 M formic acid solution. The results were analyzed using the STORM PhosphoImager. Nuclease assay In general, nuclease activities of hDna2 were measured using a standard reaction mixture (20 µl) containing 50 mM Tris–HCl, pH 7.5, 25 mM NaCl, 2 mM DTT, 0.25 mg/ml BSA, the 5′- or 3′-32 P-labeled DNA substrate, and various concentrations of MgCl2 and ATP as indicated in the figure legends. NaCl inhibited the nuclease activity (20 times inhibition at 125 mM) but 25 mM NaCl was minimally inhibitory and included in the reactions to stabilize the oligonucleotide substrates. After incubation at 37°C for 15 min, reactions were stopped with 2× denaturing termination dye (95% deionized formamide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol), and boiled for 5 min. The cleavage products were separated on a 12% sequencing gel (SequaGel, National Diagnostics) using Model S2 electrophoresis apparatus (BRL, 39 cm plate) and analyzed using the PhosphoImager. Products were quantified using the ImageQuant software on the phosphorimager, Substrate cleaved (%) is calculated as follows: Substrate cleaved (%) = (product bands)/(substrate bands + product bands) × 100. Helicase assay Helicase assays were performed with the nuclease-deficient mutant of hDna2 (D294A). The standard reaction mixtures contained 50 mM Tris–HCl, pH 7.5, 25 mM NaCl, 2 mM DTT, 0.25 mg/ml BSA, 4 mM MgCl2, 4 mM ATP and 32 P- labeled helicase substrate. After incubation at 37°C for 1 h, reactions were stopped with 5× stop solution (60 mM EDTA, 40% sucrose, 0.6% SDS, 0.25% bromophenol blue and 0.25% xylene cyanole FF). Reaction products were then
  • 11. M.Pharma (Quality Assurance) separated using 8% native polyacrylamide gels containing 0.1% SDS, and detected with PhosphoImager. hDna2 nuclease substrates All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). Oligonucleotide sequences are listed in Table 1. The locations of biotinylation are indicated as underlined nucleotides in the table. The 5′ and 3′ end labeling of oligonucleotides were performed as described previously (33). Oligonucleotides were annealed as described in the figure legends to form various structures. Flap substrate for the 5′–3′ nuclease assay were made by annealing a downstream oligonucleotide, template, and upstream oligonucleotide at molar ratio of 1:2:4. The upstream oligonucleotide was omitted to make the forked substrate.