Understanding What is oxidative stress?

Oxidative stress is caused due to an imbalance between production of reactive oxygen species (free radicals) and effectiveness of antioxidant defense. Reactive oxygen species (ROS) play a crucial role in cell signaling, however when the balance between ROS production and consumption is disrupted, it can lead to cell damage. Oxidative stress can cause damage to DNA, proteins and lipids. Reactive oxygen species are produced by electron leak from aerobic respiration by mitochondria. Enzymes like NADPH oxidases, xanthine oxidases, cytochrome P450 and other oxidases also produce ROS. There are enzymes and molecules in the body that serve as antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione which removes ROS molecules from the living system.

Reduced glutathione (L-g-glutamyl-L-cysteinylglycine), a key antioxidant present in animals, plants, fungi and bacteria provides reducing equivalents in form of free thiol groups. Glutathione exist in reduced (GSH) and oxidized (GSSG; glutathione disulphide) forms in cells and tissues, and the concentration of glutathione range from 0.5 to 10mM in animal cells. The majority (90-95 %) of glutathione exist in reduced form (GSH) in healthy cells. GSH provides reducing equivalents to antioxidant enzymes, hydroxyl radicals, ROS and is itself oxidized to GSSG; therefore GSH/GSSG ratio is critical indicator of the health of cell. During oxidative stress there is decrease in levels of GSH and increase in levels of GSSG and thus GSH/GSSG ratio decreases.

What difference between Monoclonal vs Polyclonal Antibodies?

While both monoclonal and polyclonal antibodies can be used in a wide variety of applications including Western blot, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, immunofluorescence, immunocytochemistry, Biochip technology and in the diagnosis of disease, they each have their own advantages which make them useful for different applications. To determine which type of antibodies should be used for a particular application, let us try to understand the difference between the two.

Monoclonal antibodies (mAbs) represent a population of antibodies that recognize a single epitope within an antigen. Since mAbs are produced from a single B cell in the spleen or lymph nodes of an immunized mouse, the resulting antibodies are all identical. In addition, they recognize the same epitope of a specific antigen.

However, while B cells can be used to harvest antibodies, these cells have a limited lifespan and will eventually stop producing the antibody in time. To overcome this limitation, a specific antibody-producing B cell is fused with a myeloma cell to create an immortalized B cell-myeloma hybridoma which can provide a constant supply of highly specific monoclonal antibody.

Monoclonal antibodies can be raised against many targets. Specific antibody characteristics (sensitivity requirements and cross reactivity levels) can be identified and monoclonal antibodies screened to identify any cell lines exhibiting the required characteristics.

Monoclonals can also be generated to cross react with a group of molecules. This can be quite useful in cases where there are multiple possible combinations of drugs to be tested in a patient.

Monoclonals are typically rat or mouse monoclonals, but they can also be generated from various species such as rabbit and goat.

What is Magnetic Beads for Immunoprecipitation?

The use of Protein A, Protein G or Protein A/G magnetic beads in IP has been gaining popularity due to a number of reasons. For one, studies show that magnetic beads exhibit a faster rate of protein binding, and offer reduced antibody consumption and sample loss. Magnetic beads also exhibit low nonspecific binding, and optimized IgG binding capacity. Additionally, many laboratories switched to magnetic beads since they produce cleaner, more consistent results in significantly less time.

High Binding Capacity. While agarose beads may have a porous center which significantly increases their binding capacity, magnetic beads are significantly smaller than agarose beads (1 to 4μm). This gives them an effective surface area-to-volume ratio for optimum antibody binding. In addition, magnetic beads can aggregate without the need for centrifugation, thereby increasing the yield of delicately attached protein complexes.

Reduced Antibody Consumption. Since agarose beads are porous and have high binding capacity, they require larger amounts of antibodies to produce accurate results. The antibody can be trapped inside the bead and fail to properly bind the protein of interest. When this happens, you may need to use more antibody. You wouldn't have this problem with magnetic beads since they are non-porous and antibody binding is limited to the outer surface of the bead.

Keep in mind that when the amount of antibody available for the immunoprecipitation experiment is less than sufficient to saturate the agarose beads, you can end up with particles that are only partially coated with antibodies. This can be a problem since the unsaturated portion of the beads will then be free to bind with anything that will stick. In such cases, you can expect elevated background signal due to non-specific binding of lysate components to the beads.

Reduced Sample Loss. Since magnetic beads do not require centrifugation, there is no risk of aspirating immune complexes that are bounded to the beads. This also reduces the risk of breaking weak antibody-antigen binding and the subsequent loss of target protein for a more accurate quantitation of your protein of interest and better reproducibility.

What are Ampicillin and Plasmid DNA Isolation?

While ampicillin is commonly used as a selection marker for E. coli and other bacteria during plasmid DNA isolation, protein expression and gene cloning, there are several problems that you may encounter if you are not aware of its limitations.What are these limitations and how can you avoid them? Here are some things that you definitely need to know.

There are several selectable markers that can be used to identify bacterial cells that contain a specific trait. Most of these markers are genes that confer resistance to antibiotics such as ampicillin, kanamycin, tetracycline and chloramphenicol.

By introducing a selectable marker gene into the bacterial cells, the colonies that have successfully taken up the plasmid will most likely develop a resistance against that particular antibiotic while those that do not would eventually perish.The surviving colonies can then be isolated, propagated and used for subsequent downstream experimentations.

Choosing DNA Purification in Ethanol vs. Isopropanol?

Ethanol is usually the solvent of choice when it comes to precipitating DNA out of a solution but you can also use isopropanol and basically get the same results in the end. So, why do some people use ethanol while some prefer to use isopropanol? What is the difference between the two and how do you know which solvent touse for DNA purification? Here are some things you need to know to help you choose the most suitable solvent for your experimentations.

As mentioned earlier, you can use ethanol or isopropanol in precipitating DNA from the solution and get the same end results. However, the solubility of DNA differs in each of these solvent. For the record, DNA is less soluble and falls out of the solution faster even when low concentrations of isopropanol are used but there is a tendency that the salt will co-precipitate with the DNA.On the other hand, a higher concentration of ethanol is needed to precipitate DNA from the solution but then the salts tend to stay soluble, even at lower temperatures.

What is Genome Sequence Scanning?

Genome Sequence Scanning (GSS) is a bacterial identification technology that detects sequence-specific fluorescent tags on long DNA molecules that have been extracted and purified directly from biological samples. Key to the efficacy and sensitivity of the GSS technology is the microfluidic funnel used for high-throughput stretching and scanning of long strands of single DNA molecules. In the proprietary GSS detection funnels, purified and tagged DNA molecules flow in a linear conformation at high speed past a series of lasers and optical sensors, which record the length and pattern of the labels on each DNA fragment. The labels create a barcode of the DNA in the sample, which is compared to an onboard database of barcodes to identify the serotype/strain type for the organism.

The continuous-flow microfluidic funnel is critical to the rapid throughput and strain typing of sample bacteria by GSS, and PathoGenetix physics research has worked to improve the rate and reliability of DNA stretching in order to optimize the technology’s throughput and accuracy.  PathoGenetix’s APS presentation, scheduled for Wednesday afternoon, March 5, 2014, details multiple complementary mechanisms used to maximize throughput in the GSS detection funnels, including:

Optimized funnel geometry to maximize fluid velocity while maintaining uniform stretching over the desired range of DNA lengths

Improved retention of well-stretched DNA by minimizing relaxation and hydrodynamic tumbling using constant strain rate detection channels

Normalizing DNA elasticity using sheathing-flow single molecule intercalation.

What is Agarose beads?

Agarose beads are small spherical beads of agarose gel which are commonly used in gel filtration or molecular size exclusion chromatography and biomolecular purification and immobilization. These beads act as porous gel to filter mixtures of molecules based on their individual sizes. And since these beads are easy to activate, they can also be used to bind biomolecules in a reversible or irreversible manner. In addition, their inert nature and unique internal surface area can also be activated for ligand attachment, making them the ideal basis for various affinity chromatography beads such as protein A and G, and glutathione.

Some researchers are confused whether they should use agarose or Sepharose beads for their experiments but this really doesn't matter since both refers to the same product. Sepharose is just a registered trademark for agarose beads used by GE Healthcare.

Agarose beads are available in different concentrations of agarose (2%, 4%, and 6%) that alter the separation range and bead size of the agarose beads.  2% agarose has a particle size ranging between 60-200µm while 4% and 6% have particles ranging between 45-165μm.  Agarose beads exhibit broad fractionation ranges and have high exclusion limits and negligible non-specific adsorption as well.

It is interesting to note that as agarose concentration increases, its porosity decreases. This unique characteristic increases the rigidity of the agarose chains and alters their fractionation range. This also makes them ideal for cleaning up and separating a mixture of molecules in a sample based on their individual sizes or molecular weights (MW).