Showing posts with label Proteins. Show all posts
Showing posts with label Proteins. Show all posts
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.
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.
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.
Choosing DNA Purification in Ethanol vs. Isopropanol?
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 Agarose beads?
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).
What is PathoGenetix’s Genome Sequence Scanning technology?
Under the agreement, USDA-ARS will provide PathoGenetix with genetic information and bacterial strains of E. coli and Salmonella. USDA-ARS and PathoGenetix researchers will analyze the strains, both as isolates and in mixed cultures, using PathoGenetix’s Genome Sequence Scanning technology, currently in development for commercial use as the RESOLUTION™ Microbial Genotyping System. Results of the joint analysis will assist USDA-ARS in evaluation of the RESOLUTION System as a platform for rapidly identifying pathogenic Salmonella and E. coli in food samples.
PathoGenetix’s GSS technology identifies microbial DNA from complex mixtures or from isolates, and automates the process from sample preparation through data analysis to provide actionable information in five hours. Because GSS scans microbial DNA directly from a mixed culture and does not require a pure culture, it can reduce the time, complexity, skill and cost required for molecular identification and strain typing.
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 may enable quicker decisions affecting food safety and public health.
PathoGenetix has signed an agreement with independent contract testing laboratory
The RESOLUTION Microbial Genotyping System is based on PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, a breakthrough in microbial identification with significant advantages for food safety testing. GSS works directly from complex mixtures such as enriched food samples, and automates the identification process from sample preparation to final result to provide actionable information in just five hours, days faster than identification methods currently in use in the food industry.
Q Laboratories, Inc. participated in PathoGenetix’s RESOLUTION Customer Experience Program in November, where it received an initial in-depth, hands-on review of the GSS technology. PathoGenetix’s first round of evaluations for the RESOLUTION System has focused on leading contract testing laboratories serving the food safety industry. Many food producers worldwide rely on these third party laboratories for all or part of their food quality and safety testing programs.
“We are extremely pleased to be conducting onsite evaluations of the RESOLUTION Systems by highly experienced, industry-leading food contract testing labs such as Q Laboratories,” said John Czajka, PhD, PathoGenetix’s Vice President of Business Development. “These evaluations are demonstrating the speed and accuracy of the RESOLUTION System, and providing key end-user feedback on its fit and function within high-volume laboratory workflows.”
Additionally, the U.S. Food and Drug Administration (FDA) has recently begun a nine month lease of RESOLUTION System to evaluate the System for use in public health foodborne illness outbreak investigation and response as part of a three year collaboration between the FDA and PathoGenetix.
What is PathoGenetix Delivers Bacterial Identification System?
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.
What is automated system for rapid bacterial identification
The BioNumerics® software suite (Applied Maths) was used to analyze a data set of 190 pathogenic E. coli strains from the Centers for Disease Control and Prevention (CDC). Clustering of related strains was performed using patterns generated by PFGE and whole genome sequence data included in the CDC data set, and GSS fingerprints, PathoGenetix’s proprietary technology used in the RESOLUTION™ Microbial Genotyping System. For the set of E. coli isolates tested, the analysis shows a remarkably high congruence between the GSS groupings and WGS groupings, while maintaining a good concordance with the PFGE groupings. With respect to WGS, the GSS groupings also turn out to be more discriminatory than the PFGE groupings.
The collaborative research is detailed in a poster presented yesterday at the InFORM 2013 meeting being held this week in San Antonio, Texas. InFORM meetings are designed to coordinate and enhance the work of microbiologists, epidemiologists and environmental health specialists focused on foodborne disease surveillance, outbreak detection and response. The meeting is sponsored by the CDC, the Association of Public Health Laboratories (APHL), the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS), and the Food and Drug Administration (FDA), and integrates the separate PulseNet and OutbreakNet annual meetings held in previous years.
Steps to understand How to prepare biological buffers?
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Most chemical reactions are affected by the acidity of the solution in which they occur. As such, you can maintain a constant concentration of hydrogen ions within the physiological range, manipulate a particular reaction to occur or to proceed at an appropriate rate by controlling the pH of the reaction medium through the use of the appropriate biological buffer system.
Upon the addition of a strong base such as NaOH to the buffer solution, the hydrogen ion will bind with the hydroxide ion to form water. Upon the addition of a strong acid, however, the conjugate base will simply bind with the additional hydrogen ions to form acetic acid. In each case, equilibrium can be maintained.
Note: Weak acids and bases do not dissociate completely in water but exist in solution as a mixture of dissociated and dissociated molecules.
How Do Buffers Work?
All buffers have an optimal pH range over which they can moderate the changes in hydrogen ion concentration. This is generally defined as the pKa or the negative log of the dissociation constant of the acid. The pKa can be determined by using the Henderson-Hasselbalch equation:
pH = pKa + log10 [A-]/[HA]
However, since there are acids that can lose more than one hydrogen ion (polyprotic acids), they can have multiple pKa values. If the pKa values are close together, the optimal pH range will be a continuum determined by the range of pKas.
You know Which non-radioactive assays are used to determine cell cytotoxicity and cell proliferation?
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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.
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.
What is Proteases and the Identification of Proteins
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Proteases and the Identification of Proteins
Breaking down proteins for analysis is performed through proteases, of which the most commonly used is trypsin. Proteases are designed to be sequence specific and are tailor-made to produce the best protein digestion. Alternative proteases are used for specific sequencing, whereas more versatile all-around proteases may be used throughout the process of mass spectrometry. Ideally, proteases produced for the use of mass spectrometry are able to accommodate multiple strategies for digestion and have been tested for use within mass spectrometry devices. When it comes to proteases, both the digestion time and cleavage specificity are the most important factors. Digestion time may impact the reliability of the mass spectrometry results, while cleavage will impact the separated peptides.
How Mass Spectrometry Helps in Protein Identification?
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Mass spectrometry can be used for relative quantitative proteomics: By using mass spectrometry, you do not only identify the types of proteins available in a certain sample. You also get to quantify the levels of relative proteins in the sample in a cheaper, faster and more accurate manner. While you can use it in a stand-alone manner, using it in combination with transcriptomics (The global study of gene expression at the RNA level) will definitely yield more functional information.
describe the imageIt can help in the identification of protein binding partners: Mass spectrometry can help identify the specific proteome for protein-protein interactions.
It can shed light on signal transduction pathways: Mass spectrometry can help you follow specific signal transduction pathways resulting from multiple signals and time-points without actually having to resort to running multiple western blots to do it.
It can help characterize your purified protein sample: Mass spectrometry also comes in quite handy in analyzing intact purified proteins in combination with its digested version when expressing and purifying proteins for functional and biochemical assays. In such cases, mass spectrometry can help ensure that the sequence is correct and that the modifications are in the right locations. Additionally, it can also help identify impurities in your preparation.
Mass spectrometry can help discover biomarkers: Biomarker discovery and quantification is one of the most important yet most difficult applications of proteomics. However, with the use of mass spectrometry, selective monitoring of differentiating proteins can be very much possible. As such, it may improve your chances of finding a biomarker for a particular disease, drug efficacy or drug toxicity.
Moreover, mass spectrometry can also identify drug targets from phenotype-based screens, quantify proteins for which there is no antibody and provide access to proteins in most subcellular compartments.
Understanding Sequencing and Sample Prep
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Because of its focus on healthcare investment, the conference includes a wide range of companies interested and involved in clinical diagnostics and biotechnology, so of course discussions of next generation sequencing dominate. For PathoGenetix, it was a great opportunity to network with potential investors and partners, and with more than 4000 attendees and 300 companies presenting, there certainly was a lot going on. Six presentation rooms at the Westin St. Francis ran pretty much nonstop last week Monday through Wednesday, and half-a-day on Thursday.
All in all, it was a fantastic opportunity to hear current announcements and future plans from companies across the spectrum of healthcare from hospitals and health plans to drug companies and diagnostics manufacturers.
It also was a great way for us to inform current and potential partners and investors about progress with our Genome Sequence Scanning (GSS) technology and commercialization of the RESOLUTION Microbial Genotyping System, our first application targeted to food industry testing and public health foodborne illness investigations.
What is PathoGenetix has signed Sparton for manufacturing design and pilot production
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Pre-commercial units of the RESOLUTION System are currently undergoing testing and evaluation by government and food industry partners. Earlier this month, PathoGenetix shipped a pre-commercial version of the RESOLUTION System to Marshfield Food Safety, LLC, under an agreement with the Wisconsin-based microbiology- and chemistry-testing laboratory to conduct independent testing and feedback on use of the RESOLUTION System for pathogen confirmation and identification in food industry applications.
Why is protein extraction considered to be more difficult that DNA extraction
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- Temperature. Proteins are easily denatured. As such, protein extraction should be performed at very low temperatures (usually at 4oC), especially when proteases are present in the sample. However, please keep in mind that such low temperatures may have a negative effect on your chosen purification method.
- Environmental pH. Since proteins are extremely sensitive to environmental pH changes, specific buffer conditions should be maintained at all times to protect the purity of the resulting sample. Depending on the final use of the protein, you may choose whether the pH environment should promote optimal enzyme activity, maximum purification efficacy or optimum stability.
- Purity of the solution. Keep in mind that any impurities will compromise the quality of the yield and the accuracy of subsequent experimentations. Microorganisms, proteases and heavy metal ions present in the solution may degrade or inactivate the protein and may even lead to the hydrolyzation of the protein in the sample.
- Storage temperature. The half-life of most proteins is heavily dependent on the storage temperature.
- Hydrophobicity. Different proteins require different levels of buffer hydrophobicity to achieve proper solubilization. While some types of protein require some additives to facilitate solubilization, others do not need any.
- Choice of assay. You need to determine a fast and reliable assay to use with your protein. You need to make sure that the buffers used in extracting your protein do not interfere with your chosen assay.
What role do detergents play in protein solubilization?
Detergents are commonly defined as a class of molecules that exhibit an amphipathic structure. All detergents have a hydrophilic (water-loving) polar head and a hydrophobic (water-fearing) non-polar tail. Due to their unique structure, they have the ability to form or disrupt hydrophilic-hydrophobic interactions in most biological samples. Aside from its role in protein solubilization, detergents also play an important function in the following procedures:
- cell lysis
- protein crystallization
- electrophoresis
- prevention of non-specific binding in affinity purification and immunoassay procedures
In aqueous solutions, the detergent's polar head interacts with the hydrogen bonds of the water molecules while the non-polar tail ends aggregate to form highly organized spherical structures known as micelles. The concentration at which micelles begin to form (known as the Critical Micelle Concentration or CMC) is of vital importance since it provides the researcher the precise amount of detergent that should be used to allow for complete protein solubilization.
So, how do detergents release or solubilize proteins? Here's how.
Most lipids and proteins are embedded in biological membranes which consist of amphipathic phospholipid bi-layers which have almost the same structure as the detergent micelles. While these proteins are not soluble in aqueous solutions, they can be released from the lipid bi-layer by using an appropriate detergent.
Upon the introduction of moderate amounts of biological detergents (less than the detergent's CMC) into the aqueous solution, the detergent molecules begin to disrupt the biological membrane where the proteins are embedded. However, at concentrations equal to or higher than the detergent's CMC, the lipid bi-layer breaks apart and the hydrophobic end of the detergent micelle binds with the hydrophobic end of the protein to prevent them from aggregating.
How Do Detergents Solubilize Proteins?
The amphipathic properties of the detergent molecules allows them to exhibit unique properties in aqueous solutions. The polar (hydrophilic) head groups interact with the hydrogen bonds of the water molecules and the hydrophobic tails aggregate resulting in highly organized spherical structures called micelles. At low concentrations, the detergents exist as single molecules or small aggregates and as the concentration increases micelles begin to form.
Biological membranes consist of phospholipids that are similar to detergents as they have the same amphipathic properties. The phospholipids have a charged polar head normally connected to two hydrophobic groups or tails. The phospholipids assemble as bilayers, with the hydrophobic tails between two faces of polar head groups.
For biological membranes , proteins and lipids (i.e. cholesterol) are embedded in the bilayer forming the fluid mosaic model. The proteins are held in the lipid bilayer by hydrophobic interations between the lipid tails and hydrophobic protein domains. These integral membrane proteins are not soluble in aqueous solutions as they aggregate to protect their hydrophobic domains, but are soluble in detergent solutions.
Protease inhibitors are chemical compounds used to protect protein
The reason for this is simple. Most proteases that are found in most cells and tissues belong to the serine protease group (Chymotrypsin, Kallikrein, Plasmin, Proteinase K, Thrombin and Trypsin) and they need to be rendered ineffective to ensure satisfactory protein purification yields.
PMSF works by deactivating the serine hydroxyl group and any other enzyme that contains serine in its active site through an esterification process. However, if the biological activity of such an enzyme needs to be maintained, one should consider using other types of serine protease inhibitor such as AEBSF, Aprotinin or Benzamidine.
Metalloproteases, on the other hand, are best deactivated by using EDTA that chelates the metal ions required for metalloproteases activity. Other classical lock and key inhibitors such as bestatin and other protein inhibitors such as alpha-2 macroglobulin can also be used. Other proteases may be of lesser significance but they should likewise be rendered ineffective if you want to get accurate results from your assay.
How do protease inhibitors work?
Protease inhibitors work by reversibly or irreversibly deactivating the protease present in the cell lysate by binding to the active site or by modifying its structure. Either of these actions will prevent the hydrolysis of the protein sample and allow you to store your lysate for a longer duration without being degraded in the process.
However, since there is not one chemical that can effectively deactivate each known protease, most researchers prefer to use ready-to-use "cocktails" or prepare their own mixture of several inhibitor compounds to stave off proteolysis and preserve protein extracts from degradation.
Proteolytic enzymes or proteases are so important
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:
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