Microamaze

Have You heard of the chimera of Greek mythology; part lion, part goat, part dragon, was a creature that was eventually slain by the heroBellerophon. This had its counterpart in the Makara of Hindu myth, with the body of a crocodile and the tail of a fish, also reflected in the Western astrological sign of Capricorn

Chimeras  today are no longer mythical nor part of science fiction; modern-day biology through genetic engineering has allowed for the insertion or deletion of part of the genetic code of an animal in order to change that animal’s characteristics, or its phenotype. What have not changed are the public concerns, curiosities, and anxieties about these developments.

The first transgenic mouse had the genome sequence of the Maloney leukemia virus inserted into its genome. Transgenic animals are used extensively in the study of in vivo gene function, as models for human diseases and in the production of biopharmaceuticals. 

To read more http://microamaze.blogspot.in/2016/02/bioethics-in-transgenic-animals.html

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Hello everyone , This section give the information about the different types of PUMPS used in HPLC The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range. Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). 1) Isocratic pump - Delivers constant mobile phase composition;solvent must be pre-mixed;lowest cost pump , mobile phase solvent composition remains constant with time .Best for simple separations .Often used in quality control applications that support and are in close proximity to a manufacturing process . 2) Gradient pump - Delivers variable mobile phase  composition;can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase .mobile phase solvent (“B”) composition increases with time . Best for the analysis of complex samples .Often used in method development for unknown mixtures . Linear gradients are most popular (for example, the “gradient” shown at right) Also pumps can be Classified based on Delivery systems .       • Binary gradient pump – delivers two solvent       • Quaternary gradient pump –four solvents Solvent delivery system Solvents or mobile phases must be passed via column at high pressure (1000 – 3000psi) because the particle size of stationary phase is few µ (5-10 µ).

 To continue reading   http://microamaze.blogspot.in/2016/02/different-types-of-pumps-used-in-hplc.html

HPLC is an abbreviation for High Performance Liquid Chromatography (It has also been referred to as High Pressure LC). HPLC is a separation technique that involves the injection of sample into packed tube, where individual components of the sample are moved down the packed tube with a liquid forced through the column by high pressure delivered by a pump. Mobile Phase:  Liquid Stationary Phase                                                  Separation Mechanism  

  - Solid                                                                            Adsorption    

- Liquid Layer                                                                 Partition  

 - Ion exchange  resin                                                     Ion exchange    

 - Micro porous beads                                                     Size Exclusion    

- Chemically modified resin                                               Affinity

SOLVENTS - miscible combinations of water or various organic liquids (methanol and acetonitrile), buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent

PRINCIPLE:         Analyte is forced through a column of the stationary phase by pumping a liquid  at high pressure through the column. The sample is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The time at which a specific analyte elutes is called the retention time. Pressure increases the linear velocity. Separated components are detected at the exit of this tube by a flow-through device) that measures their amount. An output from this detector is called a “liquid chromatogram”.

MAJOR HPLC COMPONENTS AND THEIR FUNCTIONS …

http://microamaze.blogspot.in/2016/02/high-performance-liquid-chromatography.html

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With love -Dixy Related Articles AGAROSE GEL ELECTROPHORESIS

Polyacrylamide Gel Electrophoresis (PAGE)

Sodium Doedecyl Sulfate 

Polyacrylamide Gel Electrophoresis (SDS PAGE)

2 D -Gel Electrophoresis 

For more information 

The study of medicines derived from natural sources.

The word "pharmacognosy " is derived from the Greek words φάρμακον pharmakon (drug), and γνῶσις gnosis (knowledge).

Defination :

It is defined as  pharmacognosy as "

The study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources"

. It is also defined as the study of crude drugs.                                                                                 OR

"The study of natural product molecules (typically secondary metabolites) that are useful for their medicinal, ecological, gustatory, or other functional properties.

History :

During 19th and  the beginning of the 20th century—"pharmacognosy" was used to define the branch of medicine or commodity sciences which deals with drugs in their crude, or unprepared, form. Crude drugs are the dried, unprepared material of plant, animal or mineral origin, used for medicine.  

As late as the beginning of the 20th century, the subject had developed mainly on the botanical side, being particularly concerned with the description and identification of drugs both in their whole state and in powder form. Such branches of pharmacognosy are still of fundamental importance, particularly for pharmacopoeial identification and quality control purposes, but rapid development in other areas has enormously expanded the subject.

Although most pharmacognostic studies focus on plants and medicines derived from plants, other types of organisms are also regarded as pharmacognostically interesting, in particular, various types of microbes (bacteria, fungi, etc.), and, recently, various marine organisms.

Fields under Pharmacognosy:

Medical ethnobotany: the study of the traditional use of plants for medicinal purposes;

Ethnopharmacology: the study of the pharmacological qualities of traditional medicinal substances;

Phytotherapy:  the medicinal use of plant extracts

Phytochemistry: the study of chemicals derived from plants (including the identification of new drug candidates derived from plant sources).

Zoopharmacognosy: the process by which animals self-medicate, by selecting and using plants, soils, and insects to treat and prevent disease.

Marine pharmacognosy: the study of chemicals derived from marine organisms.

To read more 

http://microamaze.blogspot.in/2016/01/pharmacognosy.html

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Molecular modeling encompasses all theoretical methods and computational techniques used to model or mimic the behavior of molecules.  Molecular modelling allow scientists to use computers to visualize molecules means representing molecular structures numerically and simulating their behavior with the equations of quantum and classical physics , to discover new lead compounds for drugs or to refine existing drugs in silico.

The term “ Molecular modeling “expanded over the last decades from a tool to visualize three-dimensional structures and to simulate , predict and analyze the properties and the behavior of the molecules on anatomic level to data mining and platform to organize many compounds and their properties into database and to perform virtual drug screening via 3D database screening for novel drug compounds The common feature of molecular modelling techniques is the atomistic level description of the molecular systems. This may include treating atoms as the smallest individual unit (the Molecular mechanics approach), or explicitly modeling electrons of each atom (the quantum chemistry approach).

Uses:

The techniques are used in the fields of computational chemistry, drug design, computational biology and materials science for studying molecular systems ranging from small chemical systems to large biological molecules and material assemblies. The simplest calculations can be performed by hand, but inevitably computers are required to perform molecular modelling of any reasonably sized system. Molecular modelling is a collection of (computer based) techniques for deriving, representing and manipulating the structures and reactions of molecules, and those properties that are dependent on these three dimensional structures.. The techniques described include:

        Molecular visualisation

       Methods for Equilibrium and transition state geometry location

       Introduction to Molecular mechanics methods

       Applications of Semi-empirical, ab initio and density functional molecular orbital methods

       Methods for topological analysis of wave functions

Why is molecular modeling important to the teaching of chemistry?

• Models are what we teach. Students need to learn to “think like a molecule”. To do this they need to “see” what a molecule sees and “feel” what a molecule feels. Models give us the best and most direct view of the molecular world.

• Modeling is the best tool for learning about chemical theory. VSEPR, Lewis structures, Hückel MO are all crude attempts to convert good theories into chemical predictions. Modern computational methods give a much more accurate assessment of theoretical predictions.

• Models are easy to use, inexpensive, safe. Modeling is a student-friendly educational too. It is not just for “experts”.

Applications

Molecular modelling methods are now routinely used to investigate the structure, dynamics, surface properties and thermodynamics of inorganic, biological and polymeric systems. The types of biological activity that have been investigated using molecular modelling include protein folding, enzyme catalysis, protein stability, conformational changes associated with biomolecular function, and molecular recognition of proteins, DNA, and membrane complexes.

Original article http://microamaze.blogspot.in/2016/01/molecular-modelling.html

With love 

-Dixy

Similar article

PHARMACOGNOSY

Hello everyone,      

Today I am discussing a different method for quantification of DNA ie Quantifying DNA in Solution with PicoGreen

  I have covered similar topics earlier....

DNA QUANTIFICATION USING MINIGELS

Estimating the concentration of DNA by Fluorometry using Hoechst 33258

PicoGreen dsDNAQuantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for subcloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays.

The PicoGreen assay allowed the detection of 25 pg/ml dsDNA , surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The assay is most quickly and easily performed using a handheld fluorometer.

The standard PicoGreen assay protocol is also simpler than the Hoechst 33258 method, because a single concentration of the PicoGreen Reagent allows detection over the full dynamic range of the assay.Unlike hoechst – based assay, assay using a single concentration of picogreen are linear over four orders of magnitude in DNA concentration from 1ng/ml to 1 µg/mL.

  PRINCIPLE:-

PicoGreen  dsDNA Quantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biology procedures.The brightness of this reagent is due to its high quantum yield and large molar extinction coefficient. These procedures include cDNA synthesis for library production, DNA fragment purification for sub cloning, and diagnostic applications, such as quantitating DNA amplification products,  and primer extension assays.

  REAGENTS

       Picogreen dsDNA quantitation reagent

       Reference DNA (eg calf thymus DNA) at concentration of 2 µg/mL  in TE(pH 7.5)

        TE (pH 7.5)

To read more http://microamaze.blogspot.in/2016/01/quantifying-dna-in-solution-with.html 

Other methods for DNA Quantification DNA QUANTIFICATION USING MINIGELS Estimating the concentration of DNA by Fluorometry using Hoechst 33258 Extraction and Purification of Plasmid DNA by Alkaline Lysis MethodEstimation of DNA by Diphenylamine method

With love

Hello everyone,Today I am going to write about one of the methods used to determine Concentration of DNA ie estimating the concentration of DNA by Fluorometry using Hoechst 33258. I have already covered some methods to determine concentration of DNA

AGAROSE GEL ELECTROPHORESIS, Estimation of DNA by Diphenylamine method , DNA QUANTIFICATION USING MINIGELS

INTRODUCTION:-

Quantitation of DNA is a prelude to many practices in Molecular Biology. Accurately determining the DNA concentrations of crude chromosomal or purified plasmid DNA samples is not only desirable, but an essential step in quantitative manipulations of DNA. Common techniques that use DNA, such as sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (e.g. Random prime labeling), etc., all benefit from a defined template concentration. Failure to produce results from these techniques can sometimes be attributed to an incorrect estimate of the DNA template used. DNA concentration is measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1cm path length cuvette.

PRINCIPLE:-

Hoechst 33258 (bisbenzimidazole ) is indol derived stain in free solution has maximum at 356 nm and emission maximum at 492nm . Hoechst 33258 only bind efficiently to small fragments of DNA. Concentration of Hoechst 33258 in reaction mixture should be kept low (5X10-7 M to 2.5X10-6). All DNAs and solutions should be free of ethidium bromide. Hoechst 33258 dye is cell permanent.

Fluorometry assay with Hoechst 33258 do not work at extremes of pH and are affected by both detergents and salts. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm.

The use of Hoechst 33258 in conjunction with the fluorescence microplate reader offers high specificity, as well as high sensitivity for ds DNA quantitation. The concentration of DNA in unknown sample is estimated from std curve constructed using set of std dna (0-20  µg/mL   ) whose base composition is Same as unknown.

REAGENT :-

DNA of known concentration required to construct a std curve

Florometry buffer :- 2MNaCl , 50 mM sodium phosphate (pH7.4). Prepare 500mL and srerlize through a 0.45  filter

High molecular weight DNA solution (100 µg/ml, in TE pH 8.0)

Hoechst 33258 dye (1 mg /ml in water)

Test DNA sample.

EQUIPMENT:-

Low fluorescent background

Black , U shaped ,96- Microfluor B plates

Plate reader

 To read more  http://microamaze.blogspot.in/2016/01/estimating-concentration-of-dna-by.html

With love

-Dixy

What Exactly is a chromosome?

Chromosomes are the rod-shaped , filamentous bodies present in the nucleus , which become visible during cell division . They are the carriers of the gene or unit of heredity. Chromosome are not visible in active nucleus due to their high water content , but are clearly seen during cell division.

The eukaryotes  there are 3 the usual type of chromosomes in their body cells,  but may also contain some unusual and special types of chromosomes in some body cells or at some particular stage of their life cycle. The special type of eukaryotic chromosomes are following:

1)Lamp brush chromosomes:

These are the largest known chromosomes found in the yolk rich oocytic nuclei of certain vertebrates such as fishes, amphibians, reptiles and birds.

seen with naked eye and are characterized by fine lateral loops, arising from the chromomeres, during first prophase (diplotene) of meiosis.

Germline chromosome with lat­erally projecting loops is termed as lamp brush chromosomes. This name was given because it is similar in appearance to the brushes used to clean lamp chimneys in centuries past.

These are found in , frog, toads, salamanders

The bristle are loops of unwound DNA with many RNA polymerase and mRNA molecule

In diplo- tene stage of meiosis, the yolk-rich oocytes of vertebrates con­tain the nuclei with many lamp brush-shaped chromosomes of exceptionally large sizes.

2)Giant chromosomes or salivary gland chromosomes or  Polytene chromosomes :

Found in the larvae of certain dipterans. Occur in the larval salivary glands, midgut epithelium, and rectum and Malpighian tubules of various genera (Drosophila, Sciara, Rhynchosciara, and Chironomus)

Balbiani in 1881 was first observed salivary gland chromo­somes in the salivary glands of dipteran hence it is called as salivary gland chromosomes.

Due to presence of many chromonemal fibrils on them these chromosomes are termed as polytene chromosomes by Koller.

During larval development, specific areas on polytene chromosomes become uncoiled, forming localized regions called ‘pufs’

With love

- Dixy

To read more

Hello everyone , today I am going to write about Fajan's Rule

Fajans’ rules was formulated by American chemist of Polish origin Kazimierz Fajans (1887-1975), indicating the extent to which an ionic bond has covalent character caused by polarisation of the ions. 

Covalent character is more likely if:

1. the charge of the ions is high;

2. the positive ion is small or the negative ion is large;

3. the positive ion has an outer electron configuration that is not a noble- gas configuration.

In an ionic solids there are cations and anions which are closely packed in a crystal in such a way that forces  of attraction in oppositely charged ions overcome the force of repulsion in like ions. Let  us consider 2 oppositely charged ions  X + and Y -  . Here  charge density is higher on cation ( small size) than anion , therefore electron cloud of anion is electrostatically attracted by cation. As a result the anion  Y - is polarized or deformed or distorted . The outer electron of  anion Y - are attracted in middle of X + and Y -  . It seems that electron is shared between 2 atoms , and hence imparts a slight covalent character to ionic bond . hence greater the polarization of anion , higher will be covalent character in ionic bond.

What is Fajan Rule of Polarisation?

To read more  http://microamaze.blogspot.in/2015/12/fajans-rule.html

The Golgi complex is referred to as the manufacturing and the shipping center of the eukaryoric cell. The Golgi apparatus or the Golgi body or Golgi complex or Golgi is a cellular organelle present in most of the cells of the eukaryotic organisms. Golgi apparatus was discovered in the year 1898 by an Italian biologist Camillo Golgi. It was on of the first cellular organelles to be discovered and observed in detail due to its large size. The term Golgi apparatus was used in 1910 and in 1913 it first appeared in the scientific literature

The Golgi apparatus is a series of flattened sacs that sort and package cellular materials.The Golgi apparatus has a cis face on the ER side and a trans face opposite of the ER.The trans face secretes the materials into vesicles, which then fuse with the cell membrane for release from the cell.

Function of Golgi include :

The cell synthesize a huge amount of variety of macromolecules. The main function of the Golgi apparatus is to modify, sort and package the macromolecules that are synthesized by the cells for secretion purposes or for use within the cell.

It mainly modifies the proteins that are prepared by the rough endoplasmic reticulum ( Processing of Protein).

They are also involved in the transport of lipid molecules around the cell.

They also create lysosomes.

The Golgi complex is thus referred as post office where the molecules are packaged, labelled and sent to different parts of the cell.

The enzymes in the cisternae have the ability to modify proteins by the addition of carbohydrates and phosphate by the process of glycosylation and phoshphorylation respectively.

In order to modify the proteins the golgi complex imports substances like nucleotides from the cytosol of the cell. The modifications brought about by the golgi body might  form a signal sequence. This determines the final destination of the protein.

The Golgi complex also plays an important role in the production of proteoglycans.

It is also a major site of synthesis of carbohydrates. These carbohydratres includes the synthesis of glycoasaminoglycans, Golgi attaches to these polysaccharides which then attaches to a protein produced in the endeoplasmic reticulum to form proteoglycans.

There are more functions ............ To read them follow http://microamaze.blogspot.in/2015/12/function-of-golgi-apparatus.html

With love

Hello everyone,Today I am going to write about "Mesenchymal stem cells"

First Starting  with Stem cell

Stem cells are basic cells of all multicellular organisms having the potency to differentiate into wide range of adult cells. Self renewal and totipotency are characteristic of stem cells. Stem cell technology is a rapidly developing field that combines the efforts of cell biologists, geneticists, and clinicians and offers hope of effective treatment for a variety of malignant and non-malignant diseases.    

Stem cells are broadly classified into two categories:

Embryonic stem cells (ESC) and

Adult stem cells (ASC)

Mesenchymal stem cells are  the adult cell,  plastic-adherent, fibroblast-like cells derived from bone marrow (can also be isolated from other tissues including cord blood, peripheral blood, Fallopian tube, and fetal liver and lung). Multipotent stem cells, MSCs differentiate to form adipocytes, cartilage, bone, tendons, muscle, and skin..The adult stem cells possess multipotency ( meaning they can produce more than one type of specialized cell of the body, but not all types) and differential plasticity which can be exploited for future generation of therapeutic options.

MSCs have a high capacity for self renewal while maintaining multipotency. Thus, mesenchymal stem cells have enormous therapeutic potential for tissue repair. MSCs have been shown to be capable of differentiating into multiple cell types including adipocytes, chondrocytes, osteocytes, and cardiomyocytes. Mesenchymal stem cells (MSCs) occur at low frequency in tissues and must be expanded in vitro to obtain sufficient numbers for research and therapeutic applications.

To read more http://microamaze.blogspot.in/2015/12/mesenchymal-stem-cells.html

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Viroids are infectious RNA. These are Circular RNA molecules which infect plants. Viroids are the smallest infectious pathogens known. This RNA molecule Don’t code for proteins and disrupt regulatory systems in plants. It Causes growth problems in plants. Eg Hepatitis D viroid causes Hepatitis D in humans (in combination with virus).  The human pathogen hepatitis D virus is a defective RNA virus similar to viroids .

History of Virods: Viroids, the first known representatives of a new domain of "sub-viral pathogens", were discovered, initially characterized, and named byTheodor Otto Diener, plant pathologist at the U.S Department of Agriculture's Research Center in Beltsville, Maryland, in 1971. The first viroid to be identified was Potato spindle tuber viroid (PSTVd). Some 33 species have been identified. The "RNA world fossils" hypothesis suppose that viroids were present all along the"green line" giving rise to plants, and probably before, and does not explain why they have not been found (yet) in other organisms.

VIROID DISEASES:- 1) Potato spindle tuber viroid (PSTVd)a. May be limiting to potato growersb. First viroid characterizedc. Many variants describedd. Control with detection in mother stock, clean seed 

To read more http://microamaze.blogspot.in/2015/12/viroids.html

 with love 

Hello everyone , Today I am here  with some interesting facts and Myths about  fomitic transmission of disease

So lets see what are they . First starting with some

Facts

Typical kitchen sink contains more fecal bacteria than typical flush toilet

But doorknobs and walls have fewer microorganisms

40% of sterile cloths washed in unbleached laundry picked up fecal bacteria

At high temperatures (131F) some bacteria (E.coli) get killed, however some Salmonella can survived

Toilet stall nearest the door is the cleanest. Best Bathrooms: Hospital Emergency Room and Fast Food Restaurants; Worst: Airports, Bus Stations

At a day care center upto 50% of toys are contaminated with rotavirus

You'r mobile phones can harbour various potential pathogens of zoonotic. Depressions at the area of key pad and other places make it a heaven of loads of microorganisms.

Now time for some

Myths

1) STDS can be caught from Toilet seats

- False: likely to catch diarrhea not gonorrhea

2) Ammonia and vinegar kill germs

–False: work on dirt, but not bacteria or viruses

3) Telephones and doorknobs spread viruses

– Maybe: not enough research for good conclusion; staph ear infections have been shown in teenagers

4) Plastic cutting boards are better than wood; or vice versa

–  False: Actually a tossup, either should be disinfected after use

·5) Casual contact, such as holding hands, hugging, or sitting next to someone who is HIV positive can spread disease

–  False: No, The virus doesn’t live on the skin and can’t live very long outside the body

To read similar  http://microamaze.blogspot.in/2015/12/facts-and-myths-fomitic-transmission-of.html

With love 

Hello everyone ,

Today I am  writing about "Diagnosis of viral infection in plant"

There are several consideration or questions that need to be asked to determine what diagnostic approach should be applied to a particular plant disease. For e.g.   Which virus is infecting  the plant?   What virus strain is infecting plant?   cost and the sophistication of the  technique .

Then how does a plant pathologist  go about diagnosing plant problems? The diagnostician must have very good observation skills, and s/he also needs to be a good detective. It is important to keep an open mind until all of the facts related to the problem can be collected. The possibility of multiple causal factors must also be considered.

Collection of specimen for viral testing:

    Specimen that are collected for viral diagnosis.

    Infected plant leaves

    Plant sap

     stem

The three general approaches for laboratory diagnosis of plant viruses:

1)   Direct detection:

Directly visualize virus particles contained in specimen.The morphology of the particle enables the examiner to assign the virus to a particular family of viruses. It is a rapid method.     

 Eg -Electron microscopy. PCR, IMMUNO-ELECTRON MICROSCOPY

2) Virus isolation:

3) Viral serology :

To read more  http://microamaze.blogspot.in/2015/12/diagnosis-of-viral-infection-in-plant.html

With love 

Hello everyone,This section include the method used in

White’s method

 A tomato plant that was systemically infected with TMV was used.

 It’s stem was cut into segments and washed thoroughly

These segments were suspended by threads in conical flask     containing little water

And allowed to develop roots

After some time placed in 50 ml of a nutrient broth.

The tips were allow to grow for about  a week.

Tips were cut into 1 cm and sub- cultured into fresh medium.

 The sub-cultured root tips were then removed and used for studies

 Using this technique ,it has been possible to culture other viruses such as tobacco necrosis virus and wound tumor virus.

http://microamaze.blogspot.in/2015/12/whites-method.html

With love 

-Dixy

Limulus Amebocyte Lysate (LAL) tests detect and quantify bacterial endotoxins extracted from the outer membrane of gram negative bacteria. The critical component of the LAL reagents used in endotoxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limulus Polyphemus. It contains the proteins of the blood clotting mechanism, which is triggered by endotoxins. LAL reagents are primarily used to test for endotoxins in injectable pharmaceuticals, biological products, and medical devices. They are also used in renal dialysis centers and a wide range of other applications.

Principle: In invertebrates, the amebocytes fulfil the function of white blood cells in vertebrates. They defend the organism against pathogens; therefore, they respond to the influx of endotoxins from the bacteria by releasing a series of enzymes. The amebocytes contain procoagulant enzymes (LAL enzymes ) that trigger a chain of reactions. The final product of these chain reactions is a gel comprised by coagulated proteins . If a lysate of the amebocytes extracted from the Horseshoe Crab in a watery environment is diluted, this could serve to detect very small quantities of endotoxins.

One of the main advantages of the LAL test that the crabs, from which the hemolymph is extracted to prepare the LAL reagent, remain alive. The LAL test can be conducted by using different methods to measure the process of gelation that occurs as a response from the amebocytes against the endotoxins. These methods are the so-called Gel-Clot method, turbidimetric and chromogenic methods. These  test is approved for endotoxin testing of multiple therapeutic types including cell therapy, recombinant protein, monoclonal antibody, vaccines and gene therapy. The choice comes down to whether you need quantitative results or simply to detect endotoxin in your drug.

GEL CLOT ASSAY

The Gel-Clot method is based on the presence or absence of a gel clot in your sample tube. The gelation occurs when proteins are coagulated due to the presence of endotoxins. The detection limit is normally between 0.01 and 0.03 endotoxin units per one millilitre of the solution used in the test. TURBIDIMETRIC TESTING

Microtest turbidimetric testing aims to detect endotoxin found on medical devices or in drugs. Turbidity testing determines the cloudiness of a solution by measuring the loss of intensity in a light beam passing through that solution. Turbidimetric testing’s lowest limit of detection (LLD) is 0.005 EU/mL

CHROMOGENIC LIMULUS AMEBOCYTE LYSATE

The Chromogenic Limulus Amebocyte Lysate (LAL) Test is a quantitative test for gram-negative bacterial endotoxin.  The natural substrate, coagulogen, is replaced by a chromogenic substrate. On cleavage of this substrate a chromophore is released from the chromogenic peptide and is measured by spectrophotometry. at 405-410 nm. If endotoxin is present in the sample, a yellow color will develop . The sensitivity of this assay is 0.10 EU/ml.

To read more http://microamaze.blogspot.in/2015/12/the-limulus-amebocyte-lysate-test.html

With love 

-Dixy