BIOMAG

Immunophenotyping is a most important and regularly used technique  in Immunology and Immunological research studies. The technique is usually used for the disease monitoring and diagnostic purpose either by immunohistochemistry or by utilizing the high-throughput technique Flow Cytometry.. Flow Cytometry is a common method for the immuno phenotyping the different cell subsets or sub populations of cells. Immunophenotyping is used for the identification and characterisation of the cell surface markers and intracellular markers. In simple terms, the technique is used to detect and study the proteins expressed by the cells. Samples used for the study could be Tissue Section (Fresh or Fixed) or Cell suspension (mono-dispersed cell suspension for FCM). This technique helps in rapid and easy phenotyping of each cell in a heterogeneous sample according to the presence or absence of a protein combination. Markers (or proteins of interest) are usually functional membrane proteins...

Gene Cloning Technique in Molecular Biology Field Involves the following steps: 1.a) Isolation of Genetic Material and Gene Sequence of Interest.  First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer.  Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure.  Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of...

Image Source: genomecore.ucsf.edu Real Time PCR is a molecular biology instrument which is able to monitor the amplification of the target or template DNA at each cycle. Hence the name "Real Time PCR".. It can also be called as quantitative PCR as it is able to quantify the template DNA. It works on the basis of polymerase chain reaction in addition to that it utilizes the fluorescent dyes, fluorescent reporters or probes to monitor the amplification of template in DNA by emitting the fluorescence at each cycle.  There are two main methods for the detection of amplification of DNA in real time.  1) Non-specific fluorescent dyes such as SYBR Green Dye which binds to any double-stranded DNA present in the PCR tube.  2) Sequence-specific DNA Probes consisting of oligo nucleotides which are labelled with fluorescent reporters.  Example: TaqMan. Principle of Real-Time PCR :     "A thermal cycler which is installed with an illuminator wh...

                         A PCR (Polymerase Chain Reaction) is one of the great and most widely used to technique in molecular biology. and has wide range of applications in medical and clinical laboratories for diagnosis purposes and this technique is widely and most commonly used in biological or bio medical research.  But, there are many types of PCR which have different applications and different levels of sensitivity. Some of the types of PCR are listed below with brief explanation.  Ligation-Mediated PCR : uses small DNA oligo nucleotide linkers (or adaptors) which are first ligated to DNA fragments of the target or template DNA.  Reverse Transcription PCR:  a reverse transcription enzyme called "reverse transcriptase" which will amplify the RNA strand to DNA strand called cDNA (complementary DNA). In simple terms, this type of PCR generates complementary DNA Strands from a t...

             PCR stands for "Polymerase Chain Reaction" in biological sciences, especially in molecular biology. A molecular biology technique which makes millions of copies of a target DNA. A target single copy of DNA molecule is exponentially amplified which produces millions of copies of that particular target DNA segment with the help of primers, enzymes and nucleotides.       PCR has wide range of applications in molecular biology.. This technique has been regularly used in clinical and medical laboratories, in biological or biomedical research and forensic sciences (crime investigation.  Most of PCR works on the principle of Thermal Cycling. Thermal Cycling is a process where the DNA molecules and constituents along with that segment is repeatedly heated and cooled.  Principle of PCR :                     "In the first step, the target DNA segment is heated...

Affinity chromatography was originally developed for the enzyme purification. Now, it has been extended to immunoglobulins, membrane receptors, nucleic acids, nucleotides, whole cells and cell fragments.  It is theoretically capable of giving absolute purification from complex mixtures in a single process. Affinity precipitation , in which the ligand is attached to a soluble carrier that can be subsequently precipitated by for example pH change.  Affinity Partition , in which the ligand is attached to a water-soluble polymer such as polyethylene glycol and which, with the ligand bound, partitions into an aqueous polymer phase that is in equilibrium with a pure aqueous phase.  In this chromatography, the most commonly used matrix are, cellulose, agarose, polyacrylamide, polystyrene, cross-linked dextrans and porous glass antisilica.  Applications of Affinity Chromatography :  1. used to purify a large variety of macromolecule such as ...

The tall glass columns which are packed with a synthetic ion-exchange resin is used to carry out the separation. (Resins are the polymers to which various ionizable groups have been chemically added). 

This type of chromatography is also called as "Molecular Sieve Chromatography" or "Molecular  Exclusion Chromatography". When a gel having pores on its matrix is packed in the column and a heterogeneous mixture is pass through it, smaller ions enter the gel and come out of the column phase. The bigger molecules can not enter the gel, they move only with the mobile phase through the inner phases of the gel. In this process, a type of sieving of molecules occur.     Widely used gels are Agarose, Porous Glass, Sephadex, Polyacrylamide, Polystyrene and Silica Granules.  Gel Permeation chromatography can be carried out using the technique of column chromatography. (By determining the distribution coefficient, molecular weight of macro molecules can be determined).  

Thin Layer Chromatography principle is similar to paper chromatography. The sample is spotted at one end of a coated glass plate, with the slurry of the adsorbents. Usually, Silica gel or Sodium Acetate. Using solvent systems develops the spotted plate and the spots are deducted as in paper chromatography. 

In this chromatography technique, the substance is analyzed by allowing the flow of solvents on a a specially designed filter paper. Due to capillary action, the solvent rises up on the paper and due to differential migration of the substances of a mixture, the separation is achieved.     In this type of chromatographic technique, Whatman Paper I is extensively used and this paper is available in different grades. The sample is prepared by using proper solvent and is applied on the  paper  as bands.

               An analytical technique which is used for the isolation, identification and separation of compounds based on differences in affinity for a stationary phase and a mobile phase. In chromatography, the stationary phase may be a solid, liquid, gel or a solid/liquid mixture.. and mobile phase may be a liquid or a gas. 

Electron Microscopy Image Source:  sv.rkriz.net                                           In electron microscope, the source of illumination is electron beam. The construction and principles of electron microscope are easily related to those of light microscope. The range of wavelength of visible light used in light microscope is 4000Å-7000Å, while with an electron microscope employing 60-80KV electron, the wavelength is only 0.05Å.  In the instrument, the electron gun generates electron beam. These electrons are concentrated by other components of electron gun producing a fast moving narrow beam of electron. Electrons are focused by electromagnetic lenses. Electromagnetic lens consists of wire encased in soft iron casing. When electric current is passed through the coil, it generates an electromagnetic field through which electrons are focused.  There ar...