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Friday, November 25, 2011

Principle behind the use of Salts in Hydrophobic Interaction Chromatography

The elution/precipitation strength of an ion is described by the Hofmeister series. Small, highly charged ions are strong precipitators (anti-chaotropic) whereas organic acids (eg: acetic acid) and bases have a more stabilizing effect (chaotropic) on the presence of proteins in solution. The term chaotropic refers to the ability of the ion to produce order or chaos in the water structure.


Salting in & Salting out of Protiens 

Use of Ammonium sulphate (Anti-Chaotropic agent) for binding in HIC
Anti-chaotropic salts such as ammonium sulphate and sodium sulphate expose hydrophobic patches on proteins by removing the highly structured water layer which usually covers these patches in solution. As a result hydrophobic residues on a protein molecule can interact with the hydrophobic ligands of the matrix. Salts can also reduce the solubility of proteins by shielding charged groups which normally keep proteins apart in solution. When the electrostatic charge on protein molecules are shielded, the molecules can easily interact, form aggregates and eventually precipitate (In case of hydrophobic interaction chromatography these protein molecules interact with the hydophobic ligands of the matrix). The solubility of different proteins is reduced to different extents by salt addition.

use of Acetic acid (chaotropic agent) for Elution in HIC
A denaturating agent is a chaotropic agent, but chaotropic agents aren't necessarily denaturating agents. Chaotropic agents disrupt the intermolecular forces between water molecules, allowing proteins and other macromolecules to dissolve more easily. Chaotropic agents interfere with stabilizing intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

Wednesday, November 23, 2011

Retrovirus (HIV) Replication 3D Animation

HIV Replication





  1. Fusion of the HIV cell to the host cell surface.
  2. HIV RNA, reverse transcriptase, integrase, and other viral proteins enter the host cell.
  3. Viral DNA is formed by reverse transcription.
  4. Viral DNA is transported across the nucleus and integrates into the host DNA.
  5. New viral RNA is used as genomic RNA and to make viral proteins.
  6. New viral RNA and proteins move to cell surface and a new, immature, HIV virus forms.
  7. The virus matures by protease releasing individual HIV proteins.



Courtesy: Youtube

Lecture on Gene Cloning



Video Courtesy: Youtube

Penicillin found ineffective against 40 percent of streptococcus pneumonia

A recent study on antibiotic resistance showed worrying trends of Singapore fast becoming an epicentre of antibiotic resistance with public hospitals having some of the highest rates worldwide. Further, there is suspicion that many cases of antibiotic resistance are under reported.

The rise of antibiotic resistance is an especially serious issue in Southeast Asia, as the region is a hotspot for emerging infectious diseases, including those with pandemic potential, such as severe acute respiratory syndrome (SARS), H1N1 and NDM1, an enzyme that makes bacteria resistant to a broad range of antibiotics.

This is a result of a combination of many factors, including drug resistance. Irrational drug use, frail public health systems and the wide availability of counterfeit and substandard drugs are key factors in Southeast Asia. Singapore thus faces increased risks of infectious diseases due to its proximity.

Source:

http://www.biospectrumasia.com/Content/141111SGP17469.asp


General Terms in Freeze-Drying (Lyophilization)




A Freeze - Dryer (Lyophilizer)


Accelerated Storage: Exposure of freeze dried products to elevated temperatures to accelerate the degradation process that occurs during storage.

Batch Freeze Drying: Freeze drying multiple samples of the same product in similar sized vessels at the same time in a shelf tray dryer.

Bulk Freeze Drying: Freeze drying a large sample of a single product in one vessel such as the bulk drying pans designed for
shelf tray dryers.

Collapse: A phenomenon causing collapse of the structural integrity of a freeze dried product due to too high a temperature at the drying front.

Collapse Temperature: The temperature above which collapse occurs.

Collector: A cold trap designed to condense the water vapor flowing from a product undergoing freeze drying.

Internal Collector: A collector located in the same area as the product. All water vapor has a free path to the collector.

External Collector
: A collector located outside the product area connected by a small port through which all water vapor must pass. Allows isolation of the product from the collector for drying end point determinations and easier defrosting.

Ethylene Oxide: A colorless, odorless gas used for gas sterilization of tray dryer systems.

Eutectic Temperature: The temperature at which all areas of concentrated solute are frozen.

Evaporative Cooling
: Cooling of a liquid at reduced pressures caused by loss of the latent heat of evaporation.

Freeze Drying: The process of drying a frozen product by creating conditions for sublimation of ice directly to water vapor.

Glass Transition Temperature: The temperature at which certain products go from a liquid to a vitreous solid without ice crystal formation.

Isothermal Desorption: The process of desorbing water from a freeze dried product by applying heat under vacuum.

Lyophilization: The freeze drying process.

Manifold Freeze Drying: A freeze drying process where each vessel is individually attached to a manifold port resulting in a direct path to the collector for each vessel.

Prefreezing: The process of cooling a product to below its eutectic temperature prior to freeze drying.

Pressure Gauge (Vacuum Gauge): An instrument used to measure very low pressures in a freeze drying system.

Thermocouple Gauge: A pressure gauge that measures only the condensable gases in the system. This gauge can be used as an indicator of drying end points.

McLeod Gauge: A mercury gauge used to measure total pressure in the system (i.e. condensable and non-condensable gases.)

Primary Drying: The process of removing all unbound water that has formed ice crystals in a product undergoing freeze drying.

Residual Moisture: The small amount of bound water that remains in a freeze dried product after primary drying. Residual moisture is expressed as the weight percentage of water remaining compared to the total weight of the dried product.The amount of residual moisture in a freeze dried product can be reduced during secondary drying.

Secondary Drying: The process of reducing the amount of bound water in a freeze dried product after primary drying is complete. During secondary drying, heat is applied to the product under very low pressures.

Shell Freezing: Freezing a product in a thin layer that coats the inside of the product container. Shell freezing is accomplished
by swirling or rotating the product container in a low temperature bath.

Sublimation: The conversion of water from the solid state (ice) directly to the gaseous state (water vapor) without going through the liquid state.

Vapor Pressure: The pressure of the vapor in equilibrium with the sample.

Reference:
A Guide to Freeze Drying for the Laboratory, LABCONCO, An Industry Service Publication.

Tuesday, November 22, 2011

Fundamentals of pH & Measurements - A Technical Handbook



pH Meter Image source : Google Images

A Technical handbook on pH its Fundamentals, Working, Maintenance and Applications on detail.The handbook can be obtained from the below link.

WHAT IS pH, AND HOW IS IT MEASURED?

A Technical Handbook for Industry
By Frederick J. Kohlmann

http://www.hach.com/asset-get.download-en.jsa?code=61809

Monday, November 21, 2011

CFU: Colony Forming Unit & Calculation

colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal cells. In direct microscopic counts (cell counting using haemocytometer) where all cells, dead and living, are counted,but CFU measures only viable cells. For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids. CFU can be calculated using miles and misra method, it is useful to determine the microbiological load and magnitude of infection in blood  and other samples.

cfu/ml


Example:
Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor The number of colonies per ml reported should reflect the precision of the method and should not include more than two significant figures.


Stock/ml dilutions
Serial Dilution of Bacterial Culture 


The CFU/ml can be calculated using the formula:

cfu/ml = (no. of colonies x dilution factor) / volume of culture plate

For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows:

Bacteria/ml = (130) x (10^6) = 1.3 × 10^8 or 130,000,000.

CFU/mL Practice Problems - CFU/mL Calculation Examples

Problem 1:
Five ml of Bacterial Culture is added to 45 ml of sterile diluent. From this suspension, two serial, 1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar from the last dilution. After incubation, 137 colonies are counted on the plate. Calculate CFU/mL of the original Sample?

Answer:
First thing we need to know is the Dilution Factor, or how much the original sample is diluted:
here Initially 5mL in 45mL = Final Volume / Sample volume = 50/5 = 10.

Then two serial dilutions of 1/100.

Total Dilution Factor = 10 * 100 *100 = 10^5

CFU/mL = cfu/ml = (no. of colonies x dilution factor) / volume of culture plate

= (137 * 10^5)/0.1
=1.37*10^8

So Total colony forming units = 1.37*10^8 CFU/mL

Converting CFU/mL to Log value

For example,

Total colony forming units = 1.37*10^8 CFU/mL and you want to convert it into Log value,

Just take Log(CFU/mL)

Here log (1.37*10^8) = 8.13924922.

Useful for expressing log reduction of microbes / biologic log reduction.



References
Laboratory Exercises in Microbiology

Cryopreservation of Cells and TIssues

Cryopreservation is the process of preserving cells, tissues or other substances by freezing them to very low temperatures with the help of cryogenic fluids such as Liquid Nitrogen or methanol, etc which has a lower boiling points of -196 and -96 degree Celsius.when cells and tissues are subjected to low temperature freezing the viability of cells goes which makes impossible to grow those cells after cryo-preservation to avoid this cryoprotective agents(Cryoprotectants) are used, there by increasing the survivability of the preserved samples.some of the most common cryoprotectants are DMSO (dimethyl sulfoxide), Ethylene glycol, Glycerol, 2-Methyl-2,4-pentanediol, Propylene glycol, Sucrose.
Reasons for freezing cells:
  1. Provides a continuous source of cells; unique cell lines might be impossible to replace.
  2. Genotypic drift due to genetic instability.
  3. Senescence
  4. Contamination by microbes; cross contamination with/or by other cell lines.
  5. Incubator failure
  6. Saves time and material
  7. Distribution to users
Cryoprotective Agent (CPA) / Cryoprotectant : a substance used to protect cells/tissues from damage during freezing. e.g. glycerol, DMSO
Equilibrium time: the period of time between mixing the cryoprotectants to the cell suspension in the beginning of the cooling process. (between 15-60 mins)
Living cells can be successfully preserved by freezing and can be stored for long time without much change in its viability, here cryopreservation of BHK-21 cell line is described.
Cryopreservation BHK (Baby Hamster Kidney) Cells.
AIM:
To prepare the cryoprotective medium and to subsequently cryopreserve the BHK – 21 suspension culture.
PRINCIPLE:
Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196 °C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, when vitrification solutions are not used, the cells being preserved are often damaged due to freezing during the approach to low temperatures.
PROCEDURE:
  1. Prepared 2ml of the cryo protective medium (CPM) ( 7.5% DMSO, 10% serum, rest of volume made with GMEM media of 1X concentration).
  2. Added 2.5ml of the seed and 2.5ml of CPM into a centrifuge tube and 1ml was transferred into 4 different cryovials.
  3. The remaining 1ml was used to determine cell count. The cell concentration of the seed was found to be 1.24×106 cells/ml.
  4. After 15 minutes, the cells were then subjected to gradual cooling from room temperature to 40C and 40C to -20 0C (30 min), -200C to -800C (1 hour), in different deep freezers maintained at corresponding temperatures.
  5. The vials were further transferred to the canister which was sealed with aluminium foil and eventually stored in liquid nitrogen container to cool to temperature of -176°C in vapors of nitrogen and then to liquid nitrogen at -196°C gradually.

REVIVAL OF CULTURE AFTER CRYOPERSERVATION
  1. The vials were then taken and rapidly thawed at 370C.
  2. The culture in the four vials was pooled into a centrifuge tube aseptically and add 16ml of media into it(4ml of media for 1ml of suspension).
  3. Centrifugation was done at 1200rpm for 5minutes.
  4. The supernatant was discarded and pellet was dissolved in fresh media .
  5. The cell concentration of the cryopreserved cells was found to be 1× 106cells/ml.
  6. % of Survival growth was then calculated using the formula:
Survival growth = (Post freeze count / Pre freeze count) × 100
= (1.0*10^6 / 1.24*10^6) * 100
= 80.06%
RESULT: The BHK – 21 cells were successfully cryopreserved and revived and the survival growth was found out to be 80.6 %.
This Protocol was followed during my training session on Animal Tissue Culture (BHK 21 Cell Line)  and its Preservation.

Friday, November 18, 2011

Ubiquitin: The Ubiquitous Protein and its Importance

Ubiquitin is a small regulatory protein found in the tissues of eukaryotic organisms. Ubiquitin contists of 76 amino acids and has a molecular weight of 8.5KDa. Among the 76 amino acids 7 are lysine residues. ubiquitin also has features of heat shock proteins (HSP).
Ubiquitin is a heat-stable protein that folds up into a compact globular structure. It is found throughout the cell and can exist either in free form or as part of a complex with other proteins. In the latter case, Ubiquitin is attached (conjugated) to proteins through a covalent bond between the glycine at the C-terminal end of Ubiquitin and the side chains of lysine on the proteins. Single Ubiquitin molecules can be conjugated to the lysine of these proteins, or more commonly, Ubiquitin chains can be attached. Conjugation is a process that depends on the hydrolysis of ATP.
Ubiquitin can be attached to proteins and label them for destruction. The ubiquitin tag directs proteins to the proteasome, which is a large protein complex in the cell that degrades and recycles unneeded proteins.Ubiquitin tags can also direct proteins to other locations in the cell, where they control other protein and cell mechanisms.


Ubiquitination

Ubiquitination is a post-translational modification carried out by a set of three enzymes, E1, E2 and E3. Ubiquitin is first activated by ubiquitin-activating enzyme E1, before being transferred to its active site, the amino acid cystein. This transfer requires ATP, making the process energy-dependent. The ubiquitin molecule is then passed on to the second enzyme of the complex, E2 (ubiquitin-conjugating enzyme), before reaching the final enzyme, E3, the ubiquitin protein ligase, which recognises, binds the target substrate and labels it with the ubiquitin. The process can be repeated until a short chain is formed, with three or more ubiquitin molecules usually targeting the protein to the proteasome
The tricky part of this whole process is making sure that ubiquitin is attached only to the proper proteins. Several specialized enzymes sort through the proteins in the cell and pick only the right ones. The E1 enzyme is the ubiquitin-activating enzyme that starts the process. Powered by ATP, it attaches the tail end of ubiquitin to one of its own cysteine amino acids. Then, E1 passes the activated ubiquitin to one of several E2 enzymes, the ubiquitin-conjugating enzymes, These E2 enzymes then work with a large number of different E3 enzymes to recognize obsolete proteins and attach the ubiquitin to them. The E3 enzyme is shaped like a big clamp. The target protein binds in the gap The left side of the enzyme recognizes the protein and the right side positions E2 to allow transfer of its ubiquitin.
Once an unwanted (not needed in the cell) proteins are tagged with at least four ubiquitin molecules, they are destroyed by proteasomes. Proteasomes are voracious protein shredders, but the destructive machinery is carefully protected so that it can't attack all of the normal proteins in the cell. The proteasome, is shaped like a cylinder, with its active sites sheltered inside the tube. The caps on the ends regulate entry into the destructive chamber, where the protein is chopped into pieces 3 to 23 amino acids long.
Ubiquitination and Disease Development
In certain cancers, oncogenic targets (such as the adenovirus E1 A or the oncogene c-myc) are mutated so that they are no longer subject to ubiquitination, and therefore escape degradation and accumulate in the cell. The human papilloma virus (HPV), responsible for certain forms of cervical cancer, relies on its own viral E6 protein to promote ubiquitin-mediated degradation of the tumour suppressor p53. Other cancers may promote the over-expression of E3 ligases such as mdm2 leading enhanced degradation of p53.
Ubiquitination also plays a part in diseases involving membrane proteins, such as Cystic Fibrosis, where ubiquitination is responsible for the degradation of the mis-folded CFTR chloride ion channel, or Liddle's syndrome, where a mutation in a E3 ligase (NEDD4) prevents the efficient ubiquitin-mediated degradation of the ENaC epithelial sodium channel, leading to hypertension through excessive sodium and water re-absorption.
(From Ubiquitination: labelling the proteins SEB Bulletin 2008)

Ubiquitin System Functions
The ubiquitination system functions in a wide variety of cellular processes, including:
  • Antigen processing
  • Apoptosis
  • Biogenesis of organelles
  • Cell cycle and division
  • DNA transcription and repair
  • Differentiation and development
  • Immune response and inflammation
  • Neural and muscular degeneration
  • Morphogenesis of neural networks
  • Modulation of cell surface receptors, ion channels and the secretory pathway
  • Response to stress and extracellular modulators
  • Ribosome biogenesis
  • Viral infection
The studies on ubiquitination goes beyond cancer and neurodegenerative diseases.With the rapid improvements in technologies and drug development, the relevance of these studies is tremendous. As a result, one successful anti-cancer drug has been developed and is already in use (Bortezomib, it is the first therapeutic proteasome inhibitor marketed as Velcade by Millenium Pharmaceutical), while many others are in the pipeline. Future research will be focused on specific recognition of novel target proteins by the system, with the hope to develop specific modulators that will be able to control the level of key regulatory proteins involved in basic cellular processes in health and disease.
Sources:

New Class of Anti Malarial Compound Discovered

A international team led by scientists from the Genomics Institute of the Novartis Research Foundation (GNF) and The Scripps Research Institute has discovered a family of chemical compounds that could lead to a new generation of antimalarial drugs capable of not only alleviating symptoms but also preventing the deadly disease.

Source:
http://www.sciencedaily.com/releases/2011/11/111117135722.htm