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Friday, June 29, 2012

List of Biotechnology companies in Bangalore

List of Biotechnology / Biopharma Companies in Bangalore

Aurigene Discovery Technologies Limited
29, Villa del Mar, (Near Trinity Woods)
Sarjapur Road,Bangalore-560 034

XCyton Diagnostics
15/3, Jyothi Industrial Complex, Abbigere Main Road, K. G. Halli,B`ore-15

Wipro BioMed
3rd Floor, Wipro Centre, 5,
Papanna Lane, Off.St.Marks Road,B`ore-15

Sami Labs Ltd.
19/1, 1st Main, 2nd Phase, Peenya Industrial Area, Bangalore,Karnataka,560058

Rallis Research Centre
Plot No. 21 & 22, Phase II
Peenya Industrial Area
Post Box No. 5813,B`ore-58

Millipore India Ltd.
50 A, IInd Phase, Ring Road Peenya,B`ore-58

Greenearth Biotechnologies Ltd
"Dynamic Park", 14A, Jigani industrial area

Gangagen Biotechnologies Pvt. Ltd.
6, 6th Main, BDA Industrial Suburb, Off SRS Road, Peenya, B`ore-58

Sartorius India Pvt. Ltd.
No. 10, 3rd Phase, 6th Main, Peenya
KIADB Industrial Area,
Bangalore -  560 058

Bangalore Genei Pvt. Ltd.
No.6, 6th Main, BDA Industrial Suburb0
Near SRS Road, Peenya,B`ore-58

Phalada Agro Research Foundations Pvt. Ltd.
59/10-1, 3rd Main, Gavipuram Extension,B`lore-19

Natural Remedies Pvt. Ltd.

Metahelix Life Sciences Pvt. Ltd.

Avestha Gengraine Technologies Pvt. Ltd.
Discover' 9th Floor, International Tech Park,
Whitefield Road,B`ore-66

Novozymes South Asia Pvt. Ltd.
Whitefield Road,B`ore-42

Monsanto India Ltd.
No. 277, INBRI, Phase 1, Chowdiah Road 18th
Cross Malleswaram, 560 003
Mumbai/Bangalore for Research Center

AstraZeneca India Pvt. Ltd.
After hebbal fly over,hyderabad high way,Bangalore.

Strand Life Sciences
5th Floor, Kirloskar Business Park, Bellary Road, Hebbal,
Bangalore 560024
Multiplex Bio-Tech Pvt. Ltd.
180, Ist Main Road,
mahalakshmi layout extension,Bangalore-86
Ph-3494406, 3497360,

Lifeguard Laboratories
shreya no35,12th cross,gayathri extension
Basaveswara nagar, Bangalore-79

Lotus Labs
# 201, 60 Feet Main Road, Shankarnagar, Nandini

Khoday Biotek-Palm Grove Nurseries
7th Mile, Kanakapura Road,bangalore-560 062

Vittal Mallya Scientific Research Foundation
P.B. # 406, K. R. Road,bangalore-560 004

Indo-American Hybrid Seeds (India) Pvt. Ltd.
17th Cross, 2nd A Main, Banashankari 2nd Stage,

Wexford laboratories.
91/6,1st block,nethaji road,t.r nagar,

Clintec(india) international pvt ltd,
no-104,2nd cross,ramareddy layout,Bensoncrossroad,
Bensontown, B-46.

Novonordisk  India Limited
Plot No.32, 47-50,
EPIP Area, Whiltefield,
Bangalore - 560 066

EID Parry (India) Ltd.
Research & Development Centre, 145,
Devanahalli Road, Off Old Madras Road,Bangalore 560 049
Bigtec Labs  Pvt. Ltd.
2nd Floor, Golden Heights
59th 'C' Cross,4th 'M' Block,
Bangalore 560 010

Bhat Biotech India Pvt. Ltd.
No. 11-A, 4th Cross, Veerasandra Industrial Area,
Electronics City, 561 229--Bangalore

Biocon India Limited
20th K. M. Hosur Road
Hebbagodi, Bangalore-561229

Clinigene International Pvt. Ltd
20th K. M. Hosur Road
Electronic City,Bangalore-561 229

Syngene International (P) Ltd.

20th K. M. Hosur Road
Electronic City,Bangalore-561 229

Molecular connections
#A203,blue cross chambers,infantry road,B-1.

Glaxo SmithKline (Smithkline Beecham)
No. 71, Cunningham Road, BANGALORE-560 052

Advanta India Ltd.
309, RahejaChambers, 12,Museum Road,B`ORE-560001.
CytoGenomics (P) Ltd.
3004, 12A Main, H. A. L.
2nd Stage,Bangalore 560 008. Ph-5282332, 2274950,

Accelrys Inc.
 (A subsidiary of Pharmacopeia)
'Elite House', #16, 3rd Cross,
13th 'H' Main, Doopanhalli,
HAL 2nd Stage, Bangalore - 560008

 Alfa Laval India Ltd.
2145, HAL 1st cross
1st Main, 3rd Stage
Bm Kaval, Bangalore-560075

Wipro GE Medical Systems Ltd.
A-1, Golden Enclave, Airport Road, Bangalore-17

No.3, 6th Cross, HAL 2nd Stage, Kodihalli, 
Old Airport Rd, Bengaluru, Karnataka-560038

Jubilant Biosys (p) Ltd,
Company shifted to MAHALAKSHMI LAYOUT,

Genotypic Technology Pvt. Ltd.
112/113, embassy centre,#11,crescent road,B-1

Parry Neutraceuticals Ltd
NO-706, c manipal centre,
North block47,Dickenson Road,B-42

GE Healthcare Life Sciences
18, 2nd Floor, Cunningham Road, Bangalore-560 052
Phone-2269834, 2257520

Hulimavu Horticulture Farm- Govt Of Karnataka
Hulimavu, P. O. Box No. 7648, BannerghattaRoad,B`ore-76
Phone-6582784 / 6582775 / 6584904. bus no--369c

Namdhari Seeds
119, 9, Rajarajeshwari, Rajarajeshwari Bangalore, Karnataka 560039

Kewaunee scientific corporation
143,5th sector, hsr layout,B-34.

Krishi29, CRlayout, sarakki main road, B-78.

 Icon Clinical Research
Address-102, Prestige Capri, 4 Benson Cross Road, Benson Town
Location Bangalore 560 046

Note: Please visit respective company websites to get details and contact information. If you find something wrong please corrrect it. either by mail or through comments.

Thursday, June 28, 2012

FLow Cytometery: Principle, Applications & Advantages

Flow Cytometry is a technique for identifying and sorting cells and their components (eg: DNA) by staining with a fluorescent dye and detecting the fluorescence by using laser beam illumination.
Flow cytometer is the instrument which is used for flow cytometry.

Flow Cytometry Principle
Flow cytometry involves the analysis of the fluorescence and light scatter properties of single particles (e.g. cells, nuclei, chromosomes) during their passage within a narrow, precisely defined liquid stream.

Cell sorting using Flow Cytometer
• Flow cytometry can be used to select and purify a specific subset of cells within a population
• cell sorting based on physical, biochemical and antigenic traits.

Measurable parameters in Flow Cytometry
  • Volume and morphological complexity of cells
  • Cell pigments such as chlorophyll or phycoerythrin
  • DNA (cell cycle analysis, cell kinetics, proliferation etc.)
  • RNA
  • Chromosome analysis and sorting (library construction, chromosome paint)
  • Protein expression and localization
  • Transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent proteins.
  • Cell surface antigens (Cluster of differentiation (CD) markers).
  • Intracellular antigens (various cytokines, secondary mediators etc.)
  • Nuclear antigens Measurable parameters.
  • Enzymatic activity.
  • pH, intracellular ionized calcium, magnesium, membrane potential.
  • Membrane fluidity.
  • Apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity)
  • Cell viability.
  • Monitoring electropermeabilization of cells.
  • Oxidative burst.
  • Characterising multidrug resistance (MDR) in cancer cells.
Flow Cytometry Applications:
Flow cytometry has applications in these fields
  • Molecular biology,
  • Pathology,
  • Immunology
  • Plant biology and
  • Marine biology
  • Protein engineering:
Clinical applications in flow cytometry
  • Immunophenotyping of leukemia and lymphoma
  • Platelet Function Analysis
  • Measurement of the Efficacy of Cancer Chemotherapy
  • Cell Function Analysis
  • Applications in Transfusion Medicine
  • Organ Transplantation and Hematopoietic Cell Therapy
  • Applications in Microbiology
Advantages of Flow Cytometry
  • High speed analysis.
  • Measures single cells.
  • Measures large number of cells.
  • Simultaneous analysis of multiple parameters (up to 20)
  • Identifies small sub-populations.
  • Quantification of fluorescence intensities.
  • Sorting of predefined cell populations.
Flow Cytometer Manufacturers
Flow cytometer is manufactured by many companies, some of the are:

Becton Dickinson Inc

Beckman Coulter Inc

Hudson Robotics, Inc.

Agilent Technologies

FACScan™ BD:

FACScan Flow Cytometer - Becton Dickinson

The FACScan™ system is an automated flow cytometer from Becton Dickinson. It analyzes cells as they pass through a focused laser beam one at a time in a moving fluid stream. A BD FACScan flow cytometer can be used for routine research applications, immunophenotyping, and DNA cell-cycle analysis.
The Becton Dickinson FACScan provides the high performance and quality expected from the leader in cell analysis. It is designed and optimized, as a complete system that includes hardware, software, and reagents.

Flow cytometry: principles and applications by By: Dr. Douaa Moh. Sayed
A Review and Applications of Flow Cytometry, Daniel Heller

Disontinous SDS PAGE, Two Phase SDS-PAGE, SDS PAGE Principle


SDS stands for Sodium Dodecyl Sulphate and PAGE stands for polyacrylamide gel electrophoresis.
SDS PAGE is a technique used for separating proteins with difference in molecular weights.SDS PAGE can provide information about molecular weight, purity of particular protein preparation. The advantage of this technique is that it is simple and reproducible.

For SDS PAGE Protocol clcik here

SDS PAGE Principle

SDS PAGE works by sieving effect of the polyacrylamide gel, polyacrylamide gel can be prepared with wide range of pore size by varying arcylamide - bisacrylamide ratio.SDS an anionic detergent, which binds to the protein and imparts net negative charge to the proteins. 1.4grams of SDS binds per gram of protein.Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.Ammonium per sulfate acts as initiator for polymerization and TEMED (tetramethylethylenediamine) helps in polymerization.

When electric current is applied proteins move through the gel pores based on the molecular weight it gets separated.

For SDS PAGE Protocol clcik here

Why discontinuous gel system is used? / why gel with two different pH is used?

discontinous SDS PAGE

The two-phase, discontinuous gel system used in SDS-PAGE works because there are 3 major different charged species in an SDS gel: Cl- which has the highest mobility, then the SDS-protein complexes, and finally, the glycinate, which at pH 6.8, is the slowest. When the power gets switched on, the Cl-leads the way, followed by the protein and the glycinate essentially wedges the protein between itself and the Cl-. What this does is tightly stack the proteins into a nice, tight, thin starting zone--that's why the pH 6.8 gel is called a "stacking gel." When the complexes hit the higher pH running gel (pH 8.8), the glycinate becomes more fully ionized and its mobility increases. It now overtakes the protein-SDS complexes, follows the Cl-, and the two species leave the proteins behind. The proteins keep moving towards the anode, all with the same electrical mobility, since due to the SDS, they all have identical charge per unit mass. However, without a following charged species to keep them stacked together, they are now free to start separating based on their relative sizes.

For SDS PAGE Protocol clcik here

Tuesday, June 26, 2012

Advanced Prostate Cancer Treatment Options, Detection and Treatment

Before going for the advanced prostate cancer treatment options, one need to be properly diagnosed and this can be done by blood test, Prostate Specific Antigen (PSA) is targeted in the blood test.

Prostate cancer is the formation of tumor like growth in the prostate gland, Advance Prostate cancer treatment options includes:

Advanced Prostate Cancer Treatment Options:
  1. Surgery:It is of the advanced prostate cancer treatment option, here surgically removing the tumor or the whole gland. there are side effects associated with the surgery and cannot be performed for old people and it is dependent on health of the individual.
  2. Radiation Therapy / Radio Therapy: Radiation Therapy is another method of advanced prostrate cancer treatment option, here ionizing radiations are used to kill the cancer cells, which grows rapidly and uncontrollably forming tumors.Radiation therapy works by damaging DNA of cancerous cells, ionization causes free radical formation which there by causes the damage of cells. there are problems associated with this treatment becuse even normal cells can get affected by the radiation and can turn into cancerous cells.
  3. Hormone Therapy: This is also advanced prostate cancer treatment option mainly aims at controlling the hormone (Testosterone)  which is responsible for inducing prostate cancer.Luetinizing hormone antagonist with anti-androgens are used for treatment.
  4. Chemotherapy: Chemotherapy is also an advanced prostate cancer treatment option,In chemotherapy drugs are used to treat the cancerous cells, here the drug targets actively growing cells. chemotherapy for prostate cancer is done only when the hormone therapy had stopped responding in the individual. chemotherapy will not specifically target prostate tumor cells and it can have effects all over the body (targets actively growing cells) . chemotherapy also has got side effects.The most commonly used chemotherapy drug to treat prostate cancer is docetaxel(Taxotere®). Other drugs that might be used are mitoxantrone,epiribicin,estramustine,paclitaxel.
  5. Immuno Therapy / Biological Therapy: It is one of the advanced prostate cancer option, various Immune based strategies are used to treat prostate cancer. Provenge® is an immunotherapy treatment for prostate cancer that harnesses the power of the patient's own immune system to identify and target prostate cancer cells. immuno Therapy helps identifying cancer cells as foreign cells and attacks the cancer cells.
There are certain other treatment options used for prostate cancer those include cryosurgery

Sunday, June 24, 2012

Stem Cell Therapy: Regenerative Medicine

What are stem cells and what are the types??
Stem cells are undifferentiated cells which can differentiate to perform specialized functions...There are two different types of stem cells embryonic and adult stem cells.

"Stem Cells are Regenerative Medicine"

stem cell differentiation
Stem Cell Differentiation 

Potential Applications of Stem Cell Therapy
Stem cell therapy has appilcation in these areas
  1. Brain Damage
  2. Cancer
  3. Spinal Cord Injury
  4. Heart Damage
  5. Haematopoiesis
  6. Sclerosis
  7. Diabetis
  8. Infertility
Stem Cell Therapy shown to be give promising results and there are no other alternatives in some cases, but most of the therapy procedures are in the intial phase of clinical trials and need more study to analyze the results before it can be applied in wide variety of dissease conditions.

Stem Cell Therapy for Shrinking englarged Heart
Researchers report stem cell therapy has been successfully used to shrink hearts dangerously swollen after heart attacks.

How stem cells are utilized here is, the bone marrow is having stem cells which can differentiate into a variety of here stem cells are taken from the heart patient's own bone marrow and then injected into the damaged heart. months later, the observation shows there is a significant improvement in heart performance, and within a year a significant reduction in both scar tissue and heart size.

Much more study data and trials are needed to before the treatment is made availabe to the patients.

study co-author Dr. Joshua M. Hare, a Professor of Medicine and Director of the Inter-disciplinary Stem Cell Institute at the University of Miami Miller School of Medicine said the therapy which has been under development for around ten years, was encouraging and a big step forward.

However, much more research and time is needed before the new treatment is made available to patients, three or seven years or even a decade down the road.

Researchers discussed the findings of their study in the 17th March issue of Circulation Research.

"Heart enlargement the American Heart Association (AHA) says results from various health complications like heart attack, congestive heart failure and cardiomyopathy, a form of heart muscle inflammation. Heart valve disease and high blood pressure as a result of heart muscle thickening can also contribute to heart enlargement."

Currently, only medications and / or heart transplant are the only means of reducing the increased risk of death, disability, and hospitalization accompanying an enlarged heart.



Resarch articles on enlarged-hearts-can-be-shrunk-stem-cell-therapy

SIngle Cell Protien (SCP):Process, Applications & Benefits

Single-cell protein (SCP) are dried microbial cells or total protein extracted from pure or mixed cultures of microbes  such as algae, yeasts, fungi or bacteria (grown on agricultural wastes) used as a substitute for protein-rich foods, in human and as for animal feeds.

Single cell proteins have application in animal nutrition as: Fattening calves, poultry, pigs and fish breeding.

In the foodstuffs area as: aroma carriers, vitamin carrier, emulsifying aids and to improve the nutritive value of baked products, in soups, in ready-to-serve meals, in diet recipes and

In the technical field as: paper processing, leather processing and as foam stabilizers.

Medicinal Uses of Spirulina

•Strengthen and improve immune system 
•Phycocyanins build blood cells
•Increase antiviral activity
•Exhibits anti cancer activity
• Studies showed that spirulina consumption of 4 weeks reduced serum cholestrol level in human beings by 4.5%(Henrikson 1994) and significantly reduced body weight by 1.4±0.4 kg after 4 weeks(Becker et al 1986)
• There is no changes in clinical parameters (Blood Pressure) or in biochemical variables (haematocrit, haemoglobin, blood cells, sedimentation rate) and absence of adverse effects.
• The reduction of cholesterol is partly due to high content of gamma linolenic acid in cyanobacteria(Henrikson 1994).

SCP spirulina 

Microbes as Single Cell Protein Source

Average composition of the main groups of micro-organisms (% dry weight)
Nucleic Acids
Table: Miller et al (1976)

chlorella SCP

Used as
Used commercially

Chlorella sp.
CO2 + sunlight
Yes (Japan and Taiwan)
Scenedesmus acutus
CO2 + sunlight

Spirulina maxima
CO2 + sunlight
Yes (Mexico)

Candida utilis (Torula Yeast)
1. Confectionery
Yes (U.K.), Symba process

2. Ethanol
Yes (USA)

3. Sulphite liquor
Yes (Europe, USA, Russia)
C. intermedia
Yes; Vienna process
C. krusei (+ Lactobacillus bulgarius)
Yes; Kiel process
C. lipolytica
n-alkanes (C10 - C23) + ammonia
Yes (Russia)
Kluyveromyces fragilis
Yes (France); Le Bel process
Saccharomyces cerevisiae

Chaetomium cellulolyticum
Cellulosic wastes
Fusarium graminearum
Starch hydrolysate

Yes (U.K)
Paecilomyces varioti
Sulphite liquor
Yes (Finland); Pekilo process

Brevibacterium sp.
C1 - C4 hydrocarbons
Process developed
Methylophilus methylotrophus
Yes (U.K.),

Single Cell Protein Production Process

Single cell Protein Production process follows these steps
  1. Microbial Screening
  2. Choice of Raw Materials
  3. Process Engineering and Process Optimization
  4. Technology Development
  5. Economic Consideration / Process Feasibility
  6. Safety Concerns
Microbial screening: 

Microbial Screening is the first step in Production process, suitable microbe which yields good amount of protein need to be selected. microbial strains are collected from various habitats like soil, water, air and or from other biological materials. Microbes are selected by various studies including mutagenisis and other genetic methods, some times wild types are also  used.

Choice of Raw Material :

This part is little cumbersome and one need to focus on the correct composition of carbon suppliment which yields higher biomass production in lesser time need to be analyzed. various carbon sources are like wood waste, straw, other food processing wastes are also can be tried to optimize higher biomass production.

Substrates for Single Cell Protien Production can be subdivided into three categories:

High energy sources (natural gas, n-alkanes, gas-oil, methanol, ethanol, acetic acid); 

Various wastes (molasses, sulfite waste liquor, milk, whey, fruit wastes); and

Renewable plant resources (sugar, starch, cellulose).

Process engineering:

The technical conditions of cultivation for the optimized strains are done and all metabolic pathways and cell structures will be determined.

algal culture for single cell protein
                       Algal Bioreactor

Technology Development:

Technology development is the next step where the adoption of the technical performance of the process in order to make the production ready for use on the large technical scale.

Economic factors:
Energy consumption, cost of production are the important factor while going for large scale production phase, this need to be thoroughly analyzed and an energy efficient process need to be developed or else it will end up with loss.

Safety demands & Environmental protection:

Since the SCP produced is for human consumption or for feeding animals safety of the product need to be tested. certain microbes produces toxic compounds which can have determinal effect on humans and also for the environment, so the whole process should be monitored properly.

Advantages of Single Cell Protein 
Advantages of using microbes for  Large scale production of Single cell proteins are
  1. Single cell protein high protein and low fat content.
  2. Single cell protiens are good source of vitamin.
  3. It can be produced through out the year.
  4. Generation time of microbes are less, ie, they multiply rapidly building up the biomass, more the biomass more the protein source.
  5. Protein content is very high in dried biomass upto 85%
  6. During the production of  SCP biomass, certain microbes produce usseful byproducts such as organic acids.
  7. Waste (wood waste, food processing waste, hydrocarbons, etc) can be used as a source for carbon for growing microbes there by having advantage of environmental clean up also.
  8. Doesn't require sophisticated lab setup for algae and certain other microbes.
  9. High efficiency substrate conversion. 
Disadvantages of Single Cell Protein

Even though it single cell proteins have the above mentioned advantages, it has some disadvantages also, the major problem associated with the use of single cell proteins are
  1. Many microbes produce various toxic compounds, so consumption of such toxic can have serious effect on health of humans (Food Grade SCP), or in animals (Feed).
  2. Single Cell Protein diet suppliments can pose allergic reaction.
  3. Consuming SCP, in-taking higher amount of Nucleic acids which can lead to gastrointestinal problems.
  4. Food grade SCP production are expensive due to the need to maintain high level sterility conditions in the production facility.
SCP, Shraddha Bhatt

Saturday, June 23, 2012

Ames Test : Test for Mutagenicity

Ames Test is the test for detecting chemical compounds which are suspected to be mutagens (Mutagen: agent which causes mutation). Mutagens which induce cancer is known as carcinogen. ames test is for testing the chemical is mutagen or not. Ames test was developed by Bruce Ames, hence the name of the test is known to be Ames Test.

Before developing amest test for mutagenicity, Carcinogentic / Mutagenic testing of chemicals were done by injecting the test chemical (suspected chemical) in mice and analyze for tumors. problem with this method is mutation is low frequency and low levels of mutagenicity goes undetected making insensitive. maintaining large population of mice is a expensive event.

Bruce Ames and co-workers developed a quick inexpensive test for screening mutagenicity of large number of chemical compounds using the bacterial strain Salmonella typhimurium. 

Auxotrophic strain of Salmonella typhimurium is constructed (mutant strain), which require amino acid histidine for its growth, the auxotrophic strain of bacteria would only grow in a medium which is supplemented with histidine.

Ames Test for Mutagenicity

The test is all about the reversion of auxotrophic salmonella typhimurium to prototrophic salmonella typhimurium  (prototrophic starin of bacteria: ability to synthesize all the compounds a parent bacterial strain could synthesize) in the presence of mutagen (suspected chemical compound used for the testing).small amount of histidine is supplemented just to have few cells to grow,  if the auxotrophic bacteria grows in the petri plate containing the test chemical even without supplementing histidine, then that test chemical would be mutagenic and pose threat of inducing mutation.

Certain chemicals which are not mutagenic in its original form but might turn mutagenic when the compound is metabolized, so in the test rat liver enzyme is included so the chemical can be metaboilized and its effect can be analyzed.

Friday, June 22, 2012

Cell Disruption / Cell Lysis, Methods & Applications

Cell Disruption / Cell Lysis:

Cell Disruption is the method or process for disrupting or lysing the cell inorder to release the contents out of the cell.

Cell disruption is done to release the cell contents. Cell Lysis or Disruption is done for isolating Nucleic acids (DNA / Plasmids), Proteins (Intracellular Proteins), Organelles, etc.


Cell-disruption / Cell Lysis methods include physical methods, Chemical methods and Enzymatic Methods.

Physical Methods

Manual Grinding:

Tissue is frozen and then crushed using a mortar and pestle. Minimum amount of bufer can be used while grinding. Because of the tensile strength of the cellulose and other polysaccharides constituting the cell wall, this method was the fastest and most efficient way to access plant proteins and DNA. Even for DNA isolation from liver tissue this method can be used.

Mortar & Pestle Used for manual grinding


Sonication is a physical method of cell disruption, in this method a sonicator probe is immersed into a solution containing cell suspension to disrupt, applying high frequency sound waves causes the cell to Lyse. Usually the solution will be kept in ice bath because lot of heat will be generated during the process.

A sonicator Device for cell Disruption

Liquid Homogenization:

Liquid-based homogenization is the most widely used cell-disruption technique for small volumes . Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer, Potter-Elvehjem homogenizer and French Press. A French press consists of a piston that is used to apply high pressure to a sample volume of 40 to 250 ml, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. The equipment is expensive,but the French press is often the method of choice for breaking bacterial cells mechanically.

Freeze Thaw Method:

The freeze/thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols.

Chemical Methods

Chemical agents can be used for lysing cell membranes, chemical agents acts on the cell wall of bacteria causing it to rupture the cells and release the product out. Urea and guanidium thiocyanate are widely used for cell lysis. Detergents are used in cell lysis buffers they help to solubilize membrane proteins and lipids there by causing the cell to lyse and release its contents outside.

Most widely used Detergents are:

Triton X – 100
Tween 20
Tween 80
Sodium Dodecyl Sulfate (SDS)

Enzymatic Methods:

Lysozyme, Chitinase, Pectinase

Enzymatic methods are easy and fast but cost for these methods are also high. Lysozymes are used for Bacterial cell lysis and Chitinase for Yeast cell lysis.Pectinases are used for the lysis of plant cell walls for isolating protoplast and other applications.

Lysis Buffer Composition used in Alkaline Lysis Method for Plasmid Extraction

(Tris pH 8.0, EDTA- Ethylene diamine tetra acetic acid and Glucose)
Lysozyme- optional
50mM glucose
25mM Tris-Cl (pH 8.0)
10mM EDTA (pH 8.0)

Applications of Cell-disruption
  1. Isolating intracellular Proteins.
  2. Downstream Processing.
  3. Isolating intracellular organelles.
  4. Nucleic acid isolation.

  1. Whole cell contents are released out which makes it difficult to separate out product of interest from the mixture.
  2. Cell lysis increases viscosity of the solution making it difficult to process in the further steps.
  3. Product released into harsh environment causing the product to lose stability or activity.
  4. Might cause filtration membranes to foul during the filtration process.
  5. Enzymatic cell-disruption in large scale can be expensive.
Commercially Available Cell Disruptors / Cell Lysis Buffers / Enzymes

Qsonica Ultrasonicator

CelLyticTM MT - Mammalian Tissue Lysis/Extraction Reagent - Sigma Aldrich

Glo Lysis Buffer (GLB) - Promega


Tutorial on Cell Disruption - TELIOS T. TZANNIS - RPI 1991


Thermo Scientific Technical Resource

Monday, June 4, 2012

Hot Start Taq DNA Polymerase, Types, Advantages, Commercially available Hot Start Taq

Taq DNA Polymerase is a thermostable enzyme(Molecular Weight 94 KDa) which is widely used in Polymerase chain reaction (PCR), for amplifying short stretches of DNA. Kary mullis invented Polymerase chain reaction in 1983.

Taq DNA Polymerase was first isolated from thermophilic bacteria Thermus aquaticus, inhabiting hot springs. The most significant feature of Taq polymerase is that the enzyme is active at higher temperature.

Types of PCR enzymes
  1. Taq Polymerase: Isolated from single genus of bacteria Thermus aquaticus, thermophilus, filiformis, brockianus.
  2. Pfu Polymerase:* Isolated from small group of Archea bacteriaPyrococcus furiosus, woseii, Thermococcus litoralis.
Gene encoding taq polymerase is cloned and expressed in E.coli.

The advantage of Pfu polymerase is that it has 3' – 5' exonuclease proof reading activity, it corrects nucleotide incorporation errors, which means the amplified product will have lesser error than the product amplified
used normal Taq produces blunt end PCR product. But it is expensive and slower requires more cycling time.

Before going into the details of Hot Start Taq and Hot Start PCR, let's look into the basics of Polymerase Chain Reaction and the problems associated with Taq Polymerase / non Hot Start Taq Polymerase

Polymerase Chain Reaction has 3 steps:
  1. Initial Denaturation
  2. Annealing
  3. Extension
Polymerase chain reaction (PCR) works by denaturing the double stranded DNA at higher temperature around 94oC. This step is called initial denaturation step. Due to higher temperature hydrogen bond breaks and resulting in double stranded DNA to separate out into two single strands.

Once the double stranded DNA separates out, the primes attaches to the single stranded DNA, annealing takes place at a temperature 5oC below the primer Tm (melting temperature).

Primer binding or annealing takes place during the annealing step and next step is the extension of primers by Taq polymerase at 72oC. Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. (2n ; n denotes the cycle number).

Problems associated with normal PCR / non Hot Start Taq Polymerase 

1. Specificity
2. Selectivity
3. Yield

Taq polymerase shows activity at room temperature, once the PCR mix is prepared primers can bind each other forming primer-dimer this can get amplified by the Taq polymerase which reduces the target DNA amplification.

Non-specific binding of primers to the DNA also can result in the amplification resulting in non specific PCR product formation.

Hot Start PCR:

It is a PCR technique which helps to reduce the non-specific amplification in the initial setup stages, in this technique modified Taq polymerase called Hot Start Taq Polymerase is used.

Hot Start Taq Polymerase

HotStar Taq DNA Polymerasse - Qiagen Image Source

Hot Start Taq Polymerase is a modified version of Taq DNA polymerase, Hot start Taq polymerase is formulated with heat labile monoclonal antibodies or chemical that, at room temperature, effectively neutralize DNA polymerase activity. Full enzyme activity is regained upon denaturation of the antibody or the chemical during the initial denaturation step. Using Hot start Taq DNA polymerase, hot start step can be easily incorporated into PCR protocols which are already optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions.

Types of Hot Start Taq Polymerase

1. Antibody Based Hot Start Taq
2. Chemically Modified Hot Start Taq
3. Wax Bead based Hot Start Taq
4. Sequester Primers

*Antibody Based Hot Start Taq Polymerase*

Hot Start Taq polymerase is formulated with a monoclonal antibody is bound to the active site of the enzyme taq polymerase there by preventing the catalysis through protein-protein interaction. By raising the temperature (~95oC) antibody denatures leaving the active site free.

Low activation energy is required to remove the antibody.

95oC for around 1-5 mins is enough to remove the antibody attached to the
Taq Polymerase.

*Chemically Modified Hot Start Taq Polymerase*

A small organic compound which inhibits the activity of Taq polymerase, at higher temperature the chemical compound strips off and making the taq polymerase active. Chemically modified Taq must be activated by breaking inter molecular bonds and this requires high activation energy, 10 – 15 mins at 95oC is needed to remove the chemically bound compound.

*Wax Bead Based Hot Start Taq Polymerase*

Taq Bead Hot Start Taq Polymerase is a product from promega. *Taq*Bead™ Hot Start Polymerase consists of spherical beads of wax containing Taq DNA polymerase. Taq polymerase will be available for action only when thetemperature reaches around 60oC (at this temperature wax bead melts) there
by preventing non specific amplification and primer-dimer formation.

*Sequester Primers*

It is a novel method which uses a single-stranded DNA binding protein to sequester primers at lower temperatures, making them unavailable for extension by the polymerase. Following the initial denaturation step, the binding protein is inactivated and the primers are free to participate in the amplification reaction. This method targets the primers and not the polymerase, it is portable to a variety of thermostable polymerases. The "primer-sequestration" technique effectively blocks non-specific product formation before thermal-cycling and enhances end-point and real-time PCR reactions.

Advantages of Hot Start Taq polymerase

The advantage of using Hot Start Taq polymerase is that:

- Higher yields with high specificity and sensitivity.

- Reduced / No primer dimer formation: Hot Start Taq polymerase helps to reduce the formation of primer-dimer (primers hybridize together and amplifies), which competes with the amplification of the target DNA there by reducing the target DNA amplification.

- Set up reactions at room temperature.

- Can let reactions sit at room temperature for at least 24 hours before the cycling protocol is started.

- No long initial denaturation step in cycling protocol (For antibody based Hot start Taq)

Disadvantages of Hot Start Taq polymerase:

There are few disadvantages for Hot Start Taq Polymerase

1.The antibody is from hybridoma cells which can contaminate reactions with mammalian DNA.

2.The removal of chemical blocking group on the polymerase typically requires initial denaturation times of greater than 10 minutes which causes heat-damage to DNA samples.

Commercially available Hot Start Taq Polymerase

HotStarTaq Plus DNA Polymerase, Chemically Modified, Qiagen

GoTaq® Hot Start Polymerase, Antibody Based, Promega

TaqBead™ Hot Start Polymerase, Wax Based, Promega

Platinum®Taq DNA Polymerase, Antibody Based, Life Technologies

EpiMark® Hot Start *Taq* DNA Polymerase, NEB

JumpStart™ Taq DNA Polymerase, Sigma-Aldrich

TaKaRa Ex Taq® DNA Polymerase Hot-Start, Antibody Based, Takara

HotStart-IT Taq DNA Polymerase, Sequester primers, USB

FastStart Taq DNA Polymerase, Roche Applied Science

illustra Hot Start Maser Mix, Sequester primers, GE

Ampli Taq Gold, Chemically Modified, Applied Biosystems

Cheetah HotStart Taq DNA Polymerase, Chemically Modified, Biotium Inc.

KAPATaq HotStart DNA Polymerase, Antibody Based, Kapa Biosystems


Abgene Technical Resources

Promega Technical References

Qiagen Technical Resource

NEB Technical Refernces

Genesis Biotech Inc Resources


Kapa Biosystems

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