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 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 PathoGenetix Delivers Bacterial Identification System?

PathoGenetix, Inc., developer of an automated system for rapid bacterial strain typing, announced today that it has delivered and installed an early commercial version of the RESOLUTION Microbial Genotyping System to MRIGlobal, an independent contract research organization. MRIGlobal has purchased the RESOLUTION System as part of a U.S. government-funded project, and will be evaluating use of the System for identification and strain typing of specific organisms using MRI-developed assays.

The RESOLUTION System is based on PathoGenetix’s proprietary Genome Sequence Scanning (GSS) technology, which enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as environmental, clinical and enriched food samples. Initially developed to detect bio-threat pathogens in environmental samples under a five-year, $50-million contract through the Department of Homeland Security, the breakthrough GSS technology isolates and analyzes DNA direct from complex mixtures—without the need for a pure culture isolate. The strain type information provided by GSS is comparable in resolution to pulsed field gel electrophoresis (PFGE), one of the current gold standards for pathogen identification.

MRIGlobal and PathoGenetix have collaborated for several years in the ongoing evaluation of the GSS technology for biodefense applications. MRIGlobal has purchased a RESOLUTION System as part of a U.S. government-funded project, and will be evaluating the system for use in the rapid identification of specific organisms, using MRIGlobal-developed assays. The RESOLUTION instrument will be used to build a database for identification of select microorganisms. As one of the nation’s leading research institutes, MRIGlobal conducts programs in the areas of national security and defense, life sciences, energy and the environment, agriculture and food safety, and engineering and infrastructure.

You know Which non-radioactive assays are used to determine cell cytotoxicity and cell proliferation?

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While there are a number of non-radioactive assays that can help determine cell proliferation and viability as well as cell toxicity, determining the most appropriate assay to use can be very vital in obtaining the right information from the given cell sample. So, how do you know which one to use in each particular case? Here is a guide that can help you accomplish this task.

Measuring Cell Viability, Proliferation and Cytotoxicity

Cell viability and cytoxicity assays are best used in determining the metabolic and proliferative activities of cells within a given sample. As such, they can be used to obtain vital information on the effects of certain experimental stimulus on the proliferation of cells within an in vitro environment.

There are a number of ways by which you can get an accurate indication of the cell vitality within your sample. You can choose to assess plasma membrane integrity, mitochondrial activity and/or metabolic activity to accomplish your purpose.

Membrane integrity can be assessed by monitoring the passage of LDH to the extracellular environment. Since LDH is normally confined inside the cell, its presence in the extracellular environment indicates loss of cell membrane integrity, an occurrence that usually follows apoptosis or necrosis. If you want to measure the amount of LDH in your sample, consider using CytoScan LDH Cytotoxicity and Cytoscan Fluoro Cytotoxoicity Assays.

On the other hand, you may want to use Cytoscan WST-1 Cell Proliferation Assay if you are interested in determining cell cycle regulatory factors. This particular colorimetric assay works on the basic principle that tetrazolium salt WST-1 will be reduced to water-soluble formazan by the action of cellular dehydrogenases. The resulting formazan dye can then be accurately measured by absorbance.

For cell density determination, you can use the Cytoscan SRB Cytotoxicity Assay. This particular assay is based on the quantitative staining of cells with the fluorescent dye Sulforhodamine B, an anionic aminoxanthene dye that forms an electrostatic complex with the basic amino acid residues of proteins under moderately acid conditions. This reaction provides a sensitive linear response that can be readily measured at absorbances between 560 and 580 mm. This method is regarded to be a highly efficient and cost-effective method for screening and has a sensitivity that is comparable to those using fluorimetric methods.

Cytotoxicity Assays, you know How Are They Classified?

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Subjecting cells to cytotoxic compounds may bring different results. Cells may stop growing and dividing actively, lose cell membrane integrity and suffer instantaneous death (necrosis) or initiate a series of events that will lead to programmed cell death (apoptosis). To better understand the effects of toxic compounds on living cells and to measure the level by which they begin to exhibit their harmful effects on biological systems, researchers and pharmaceutical laboratories use cytotoxicity assays to learn what they need to know.

In general, there are three distinct types of cytotoxicity assays. There are assays that determine cell viability by:

Exhibiting a change in the membrane permeability or metabolism (viability assays);

Measuring their absolute long term survival rate and their capacity to regenerate (long term survival assays);

Exhibiting survival in an altered or genetically mutated state (irritancy assays).

Viability Assays

Can mass spectrometry help in protein analysis and identification?

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Mass spectrometry (also known as 'mass spec' or MS) is one of the most important tools used in the study of proteins. By employing a variety of mass spectrometry techniques, researchers can accurately identify and quantitate proteins in a given solution, identify amino acid sequences, and determine the overall structure of your protein of interest.

Mass spectrometry is extensively used in the field of proteomics since it provides highly accurate molecular weight information on intact protein molecules and peptides produced by enzymatic or chemical treatment of the protein sample. In addition, the fragment ions generated through mass spectrometry via collision-induced dissociation (CID) can provide accurate information on the primary structure and modifications of your protein of interest. As such, the methodical use of mass spectrometry tools can significantly improve the analysis of your samples.

Mass spectrometry is basically an analytic technique that determines the relative masses of molecular ions and fragments. Using this process, the gas phase molecules are ionized to determine their mass-to-charge ratio. Since lighter ions will travel faster and be detected first when an electric field is applied, the relative mass can be accurately measured and the composition of the molecule can then be identified. In addition, the sequence of component amino acids can also be identified using the same procedure.

Understanding Rapid Identification of Multiple Salmonella Serovars in Food Samples

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PathoGenetix™, Inc., a commercial-stage developer of an automated system for rapid bacterial identification, will present new research today demonstrating the use of Genome Sequence Scanning™ (GSS™) technology to confirm and identify multiple serovars of Salmonella in enriched food samples in less than five hours. The data, included in a poster presentation at the 4th American Society for Microbiology (ASM) Conference on Salmonella in Boston, add to a growing body of research demonstrating the use of PathoGenetix’s proprietary genotyping technology to reliably identify pathogens of public health and food safety significance, including Salmonella and Shiga toxin-producing E. coli (STECs).

The study evaluated the use of GSS in molecular serotyping and sub-typing of Salmonella, and as a tool for simultaneous detection of multiple serovars of Salmonella in complex mixtures.

Because Genome Sequence Scanning is culture independent, and fully automated from sample preparation to final report, the technology greatly reduces the time, complexity and skill required when compared to other molecular and next generation sequencing (NGS) identification approaches. The strain-type information provided by GSS is comparable to pulsed field gel electrophoresis (PFGE), the current standard for pathogen typing in foodborne outbreak investigation and response. As a result, GSS offers a powerful new tool for epidemiological investigations and outbreak monitoring that can enable quicker decisions affecting food safety and public health. The GSS technology will be commercially available in 2014 in the RESOLUTION™ Microbial Genotyping System.

According to the American Society of Microbiology, Salmonella infections continue to be a major public health problem in many parts of the world. In the U.S., Salmonella is the leading cause of foodborne illnesses leading to hospitalization and death. The Salmonella genus has more than 2,500 serotypes or serovars, based on the antigens that the organism presents on its surface. In the U.S., Salmonella Enteritidis and Salmonella Typhimurium are the most common serotypes, accounting for half of all Salmonella infections in people.

Proteolytic enzymes or proteases are so important

The study of proteolytic enzymes or proteases has always been an important component of protein research. Since these enzymes play a vital role in various biological processes such as reproduction, pre- and post-natal development and immune response, and in the development of cancer and most autoimmune, degenerative and infectious diseases, determining their specific function is considered to be of great importance in the field of proteomics. To better understand how these enzymes work, a number of protease assays are currently being used by researchers to determine their specific actions.

The total protease activity in any given protein sample can be determined by subjecting it to a protease assay kit that uses a highly quenched dye-labelled protein substrate. Upon contact, the protease present in the sample will digest the substrate and release dye-labelled peptides in the process. Absorbance is then measured to determine protease activity. In general the more label released, the higher the signal will be.

The resulting absorbance values are then compared to a standard curve. In a nutshell, a standard curve is generated by allowing various known quantities of tyrosine to react with the reagent. To accurately measure the amount of tyrosine liberated in the process, you should create the standard curve by recording the absorbance values for the standards, the test samples and the blanks (for both standard and test samples) and calculate the difference between the absorbance of the test sample and the test blank. You can compute for the activity of enzyme in units per/ml by using the following equation:

Understanding What assay development accessories should be used for protein estimation?

Now that we have covered biotin-binding and protein/peptide binding plates, it's about time we talk about antibody binding plates. So, what are antibody-binding plates and what are their most common applications? Find out what you need to know by reading the rest of this article.

Species-specific binding plates are activated plates designed to bind specifically with mouse or rabbit IgG antibodies. These plates are suitable for working with low quantities of antibodies or antibodies that are easily denatured upon direct binding to polystyrene plates, and can be used for direct, indirect, competitive and sandwich assays. Since these plates exhibit specificity to IgG, purified antibodies are not essential for the success of your research.
Plates that are designed to bind constant (Fc) domains of antibodies offer maximum exposure of the
binding site by ensuring that the antigen binding domain of the antibody is oriented away from the plate. These plates are coated with protein A/G which contains four binding sites from protein A and two from protein G to ensure maximum range of specificity and binding capacity.

Plates that are designed to bind constant (Fc) domains of antibodies offer maximum exposure of the
binding site by ensuring that the antigen binding domain of the antibody is oriented away from the plate. These plates are coated with protein A/G which contains four binding sites from protein A and two from protein G to ensure maximum range of specificity and binding capacity.

Due to the unique orientation of the immunoglobulin in these plates, their antibody capacity is far more superior as compared to plates that are directly coated with antibodies. However, please take note that the plates are suitable for single antibody assays and not for multiple assays or sandwich ELISAs.

There are also some plates that are designed to bind the kappa light chains of immunoglobulins without interfering with the antigen binding site. These protein L coated plates bind to all classes of IgG (including IgG, IgM, IgA, IgE and IgD) and also bind to single chain variable fragments (scFv and Fab fragments). They only bind kappa I, III and IV in human and kappa I in mouse, have weak binding affinity for rabbit immunoglobulins and have no binding affinity for bovine, goat or sheep immunoglobulins. These plates are pre-blocked to reduce non-specific binding capability and are designed for single antibody assays.

Understanding Targeted Sequencing Next-Generation Sequencing

Since its launch in 2012, Ion AmpliSeq targeted sequencing technology has seen broad global adoption and resulted in the publication of greater than 140 peer-reviewed publications. Applications span such studies as investigating phenovariance in mice1, assessing gene copy number alterations2, and defining genetic changes associated with lung cancer3 research.

Researchers are not only using Ion AmpliSeq technology for less challenging research samples where available sample quantity and quality are not limiting, such as cell culture or fresh-frozen tissues, but also seeing tremendous success with challenging sample types, such as formalin-fixed paraffin-embedded (FFPE) tissue, achieved samples from fine needle aspirates, and cell-free DNA extracted from blood (cfDNA).

Analysis of FFPE tumor samples
Formalin-fixed paraffin-embedded (FFPE) samples are challenging to work with. Dr. Nicole Pfarr discubrsses her experience with Ion AmpliSeq Panels on the Ion PGM System. (They currently use eight panels, both standard and custom).