Biochemical Lab Methods, Systematic and Analysis Methods

The branch of science concerned with the chemical and physico-chemical process and substances which occur in living organisms.

Biochemical analysis techniques refer to a set of methods, assay, and procedures that enable scientists to study the substances found in living organisms and the chemical reactions essential life processes. The most complicated of these techniques are reserved for specialty research and diagnostic laboratories, while simplified sets of these techniques are used in such common procedures as testing for banned drug abuse in competitive athletic events and monitor of blood sugar by diabetic patients.

List of biochemical techniques:

Biochemical lab methods, systematic and analysis methods.

• Spectroscopic methods
• Electrophoretic techniques
• Chromatography
• Calorimeter
• Photometery
• Nuclear magnetic resonance
• Centrifugation
• ELISA
• DNA cloning and sequencing
• use of radioisotopes
• immunoassay methods

1. Chromatography:

Chromatography is one of the most helpful and accepted tools of biochemistry. It is an analytical technique dealing with the separation of closely associated compounds from a mixture. These consist of proteins, peptides, amino acids, lipids, carbohydrates, vitamins and drugs.

Principles and categorization

Chromatography usually consists of a mobile phase and a stationary phase.

• Mobile phase: The mobile phase refers to the combination of substances(to be seperated), dissolve in a liquid or a gas.
• Stationary phase: The stationary stage is a porous solid matrix through which the sample contained in the mobile phase percolates. The contact between the mobile and stationary phases results in the separation of the compounds from the mixture. These interactions consist of the physicochemical principles for example adsorption, partition, ion-exchange, molecular sieving and affinity.

The interaction between stationary phase and mobile phase is frequently employed in the classification chromatography e.g. partition, adsorbtion, ion- exchange. More, the categorization of chromato¬graphy is also base either on the nature of the stationary phase (paper, thin layer ,column), or on the nature of both mobile and stationary phases (gas- liquid chromatography).

The movement of charged particles (ions) in an electric field resulting in their migration towards the oppositely charged electrode is called as electrophoresis. Molecules with a net positive charge (cations) move towards the negative cathode whereas those with net negative charge (anions) transfer towards positive anode. Electrophoresis is a widely used analytical method for the separation of biological molecules for example plasma proteins, lipoproteins and immunoglobulin’s.

Types of electrophoresis:

• Zone of electrophoresi
• Immunoelectrophoresis:
• Isoelectric focusing:

Photometry broadly deals with the learn of the phenomenon of light absorption by particles in solution. The specificity of a compound to take up light at a particular wavelength (monochromatic light) is exploited in the laboratory for quantitative measurements.

Colorimeter:

Colorimeter (or photoelectric colorimeter) is the tool used for the measurement of coloured substances. This apparatus is operative in the observable range (400-800 nm) of the electromagnetic spectrum of light. The functioning of colorimeter is based on the principle of Beer-Lambert law.

The colorimeter, in general contain light source, filter sample holder and detector with display (meter or digital). A string lamp usually serves as a light source. The filters allow the passage of a minute range of wave length as incident light.

The spectrophotometer primarily differ from colorimeter by covering the ultraviolet region (200- 400 nm) of the electromagnetic spectrum. additional the spectrophotometer is more complicated with numerous additional devices that eventually raise the sensitivity of its operation several fold when compare to a colorimeter.

A precisely selected wavelength ( 234 nm or 610 nm) in both ultra violet and visible range can be use for measurements. In place of glass cuvettes (in colorimeter), quartz cells are used in a spectro-photometer. The spectrophotometer has similar basic parts describe for a colorimeter

Ultracentrifugation is an indispensable instrument for the isolation of subcellular organelles, proteins and nucleic acids. as well, this technique is also in use for the purpose of molecular weights of macromolecules. The rate at which the sedimentation occur in ultracentrifugation primarily based on the mass and shape of the particles or macromolecules (i.e. on the molecular weight). It is expressed in terms of sedimentation coefficients).

Centrifugation is the use of the centrifugal forces generated in a spinning rotor to divide biological particles, it includes cells, viruses, sub-cellular organelles, macromolecules (principally proteins and nucleic acids) and macromolecular complexes (such as ribonucleoproteins and lipoproteins). The three main procedures of separation are differential pelleting, rate-zonal centrifugation and isopycnic centrifugation. The first two methods separate particles primarily on the basis of volume while isopycnic centrifugation separates particle on the basis of their density. The choice of centrifugation technique based on the nature of the particles and often above one separation technique is compulsory for example, membrane fractionation often involves first making an enriched fraction from a cell homogenate by differential pelleting followed by isopycnic

Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry method used in quality control and research for determining the content and purity of a test with its molecular structure. Centrifugation to attain purified fractions.

Mass spectrometry is a useful analytical technique used to quantify known materials, to identify unidentified compounds in a sample, and to elucidate the structure and chemical properties of dissimilar molecules. The whole process includes the change of the sample into gaseous ions, with or not including fragmentation, which are then characterize by their mass to charge ratios (m/z) and relative abundances.

This method mainly studies the effect of ionizing energy on molecules. It based upon chemical reactions in the gas phase wherein sample molecules are consumed during the formation of ionic and neutral species.

Principle:

The initial step in the mass spectrometric analysis of compounds is the manufacturing of gas phase ions of the compound, mostly by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. The ions are separated in the mass spectrometer in accordance with their mass-to-charge ratio, and are detected in proportion to their great quantity A mass spectrum of the molecule is thus formed. It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio. Ions give information concerning the nature and the structure of their precursor molecule. In the spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z (followed by ions containing heavier isotopes) and gives the molecular mass of the compound.

ELISA is depend on the immunochemical principles of antigen-antibody effect

1. The antibody in opposition to the protein to be determined is set on an inert solid for example polystyrene.
2. The biological sample contains the protein to be estimated is useful on the antibody coated surface.
3. The protein antibody complex is then reacted with a second protein specific antibody to which an enzyme is covalently related. These enzymes must be easily assayable and make preferably coloured products. Peroxidase, amylase and alkaline phosphatase are normally used.
4. Later than washing the unbound antibody linked enzyme, the enzyme bound to the second antibody complex is assayed.
5. The enzyme activity is identifies by its action on a substrate to form a product (usually coloured). This is rela ted to the concentration of the protein being estimated.

Applications:

ELISA is widely used for the determination of small quantities of proteins (hormones, antigens, antibodies) and other biological substances. The most commonly used pregnancy test for the detection of human chorionic gonadotropin (hCG) in urine is based on ELISA. By this test, pregnancy can be detected within few days after conception. ELISA is also useful for the diagnosis of AIDS.