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 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.

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).

What is PathoGenetix’s Genome Sequence Scanning technology?

PathoGenetix and the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS) have agreed to collaborate on an evaluation of PathoGenetix’s Genome Sequence Scanning technology for use in identifying strains of Shigatoxin-producing E. coli (STECs) and Salmonella enterica, two types of pathogens frequently implicated in foodborne illness outbreaks. The ability to quickly and accurately identify these pathogens, particularly in their most virulent forms, can have significant public health and financial impact for consumers, farmers and ranchers, and the agricultural and food industries.

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

PathoGenetix has signed an agreement with independent contract testing laboratory, Q Laboratories, Inc., to conduct an independent evaluation of the RESOLUTION™ Microbial Genotyping System. Q Laboratories will begin testing the RESOLUTION System in its Cincinnati, Ohio laboratory in April, and provide feedback on ease-of-use, speed and performance in food safety testing

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 automated system for rapid bacterial identification

PathoGenetix, Inc., a developer of an automated system for rapid bacterial identification, and Applied Maths, NV, a leader in bioinformatics and analytical solutions for public health and research laboratories, presented a novel study at InFORM 2013 comparing three different genomic methods for typing bacterial pathogens: the new technologies, whole genome sequencing (WGS) and Genome Sequence Scanning™ (GSS™), and the traditional pulsed field gel electrophoresis (PFGE). PFGE is the current standard for pathogen identification in foodborne illness outbreak investigation and response.

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 RESOLUTION System can work from a mixed sample and does not require the preparation of a cultured isolate, as is the case with whole genome sequencing and PFGE, and provides strain type and serotype results in less than five hours.

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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Buffers are essential to life. They help maintain the proper functioning of cellular systems by resisting rapid changes in pH. In living organisms, the pH of the blood is maintained at a pH of 7.4. A slightly more basic pH (7.7) would result in convulsions and muscle spasms while a slightly acidic pH (6.95) would result in coma and even death.

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.

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

Few reason Why Is Lactate Dehydrogenase (LDH) Release A Good Measure For Cytotoxicity?

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When treated with a cytotoxic compound, living cells may face one of two fates. They could either stop growing and dividing, or die through either of two distinct processes - necrosis or apoptosis. Basically, cells undergoing necrosis (accidental cell death) swell and lose membrane integrity before shutting down and releasing their intracellular contents into the surrounding environment. This type of cell death is usually triggered by external factors such as toxic chemical or traumatic physical events.

On the other hand, cells undergoing apoptosis (normal or programmed cell death) go through a series of well-defined events such as the shrinking of the cytoplasm, cleavage of DNA into smaller fragments, etc. before being engulfed by white blood cells.

When the cell membranes are compromised or damaged in any way, lactate dehydrogenase (LDH), a soluble yet stable enzyme found inside every living cell, is released into the surrounding extracellular space. Since this only happens when cell membrane integrity is compromised, the presence of this enzyme in the culture medium can be used as a cell death marker. The relative amounts of live and dead cells within the medium can then be quantitated by measuring the amount of released LDH using a colorimetric or fluorometric LDH cytotoxicity assay.

Other enzymes such as adenylate kinase and glucose-6-phosphate may also be used to measure cytotoxicity but these enzymes are not stable and lose their activity during cell death assays.

What is Proteases and the Identification of Proteins

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Protein analysis and identification through mass spectrometry first requires a breakdown of each protein into their composite peptides. Once the protein has been broken down, the peptides can be separated through the use of a reverse phase column and the peptides and peptide fragments can be measured using a mass spectrometer.

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.

Proteases are produced in-solution, in-gel, and isolated as standalone proteases, to be more useful and convenient within a laboratory setting. Pro-teases that are provided in-solution are generally preferred for smaller samples, whereas in-gel suspensions are designed for more complex operations that may require additional control. Either way, the quality of the proteases are still equally validated, and the results of the proteases should be consistent.

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.

How Mass Spectrometry Helps in Protein Identification?

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There are a lot of ways by which protein identification and research can be aided by mass spectrometry. Consider the following:

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 map out protein post translational modifications: By using mass spectrometry, you can identify and localize any modifications in your protein sample. You can pinpoint with great accuracy where your protein has been modified and identify the nature of modification as well.

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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PathoGenetix CEO Ann Merrifield and I are just back from the J.P. Morgan Healthcare Conference held last week in San Francisco. Now in its 32nd year, this annual meeting is the premier investor conference for companies in the healthcare, life science and diagnostics industries, and a unique, opportunity for investors and industry leaders to meet in one setting.

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.

Along with excitement about the system as a whole, we’re seeing some real interest in the sample preparation component of the GSS technology, both in terms of its capability and automation. Called the Genome Processor, the sample preparation component extracts and purifies genomic DNA while maintaining the integrity of long DNA fragments.

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.

What is PathoGenetix has signed Sparton for manufacturing design and pilot production

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PathoGenetix announced today that it has signed Sparton Corporation as the design and manufacturing partner for the RESOLUTION™ Microbial Genotyping System. Under the terms of the agreement, Sparton will conduct design for manufacture and pilot production of a limited number of pre-commercial instruments for the RESOLUTION System, which is slated for commercial availability in 4Q2014.

The RESOLUTION System is the first commercial application of PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, and has been developed for food safety testing in both the food industry and in public health foodborne illness outbreak investigations. The RESOLUTION System enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as enriched food and clinical samples. The bacterial strain information provided by the RESOLUTION System is comparable to pulsed field gel electrophoresis (PFGE), the current gold standard for pathogen typing in foodborne illness outbreak investigation and response.

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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While protein extraction and DNA extraction both serve as the starting point for subsequent downstream experimentations, the former is considerably more difficult to perform as compared to the latter for a number of reasons. Here are some of the factors that you may need to consider when extracting protein from your samples.

• 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 structure of detergents is key to its ability to function as a solubilization agent. Detergent molecules contain a polar head group from which extends a long hydrophobic carbon tail.

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.

A wide range of detergents are routinely used to release, or solubilize, proteins from lipid membranes. 
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.