wiki.Alumni.NET - Your Location Information Resource

From wiki.Alumni.NET

Current revision

[protein aqua strategy]http://www.creative-proteomics.com/services/absolute-quantification-aqua.htm Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e. g. , phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and post-translationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer.

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. Besides the measurements of protein expression accomplished through differential incorporation of stable isotopes into cellular proteins, the absolute quantification is a useful method in proteomics analysis.

The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modification. AQUA provides absolute quantification by employing synthetic peptides containing stable isotopes.

The absolute quantification method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than 10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested.

protein identification by peptide mass fingerprinting

Creative Proteomics provides [protein identification by peptide mass fingerprinting]http://www.creative-proteomics.com/services/peptide-mass-fingerprinting-pmf.htm (PMF) analysis and ions searching against database for rapid identification of proteins.

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification. Basically, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. Then these masses are compared to either a database containing known protein sequences or even the genome sequence which can be translated into proteins through computer programs. Then the absolute masses of the peptides from each protein are calculated theoretically for mass comparison between the peptides of the unknown protein and the theoretical peptide masses of each protein to find the best match.

glycan analysis of therapeutic glycoproteins

As one of the most important post-translational modifications of proteins in eukaryotic cells, protein glycosylation is involved in a wide range of biological and physiological processes, including recognition & regulatory functions, cellular communication, gene expression, cellular immunity, growth and development.

[glycan analysis of therapeutic glycoproteins]http://www.creative-proteomics.com/application/n-glycan-analysis.htm functions are usually determined by the structures of the oligosaccharides, which are covalently attached to proteins primarily at 2 structural motifs: the amide group of an asparagine (N-glycans) or the hydroxyl group on serine or threonine (O-glycans). Because of the diversity of the oligosaccharides, even glycosylation at a single site can generate considerable heterogeneity of the mass and charge of glycoproteins. Although different approaches for N-glycans analysis have been described, usually these methods are based on enzymatic release of N-glycans from the protein by PNGase F, and derivation of released glycans, due to the lack of intrinsic chromophores, with a fluorescent labelling before analysis.

mass spectrometry protein identification

Among the endoproteases which could be used for protein digestion, the serine protease trypsin is most commonly employed as it generates peptides which are highly amenable to MS(/MS) analysis.

Sample preparation is critical for proteomics analysis, and Creative Proteomics provides sample preparation service according to your needs.

A pure protein is one that is free from any quantifiable amounts of impurities. Any purity determination is only as reliable as the analytical methods used, and factors such as the structural properties of the protein itself, the amount of protein available, the nature of potential contaminants in the sample, and the accuracy of the estimate required should always be considered when selecting the method of analysis.

Creative Proteomics provides Peptide Mass Fingerprinting ([mass spectrometry protein identification]http://www.creative-proteomics.com/services/protein-identification.htm) analysis and ions searching against database for rapid identification of proteins.

transfected cells

Stable [transfected cells]http://www.creative-biogene.com/Product/Transfected-Stable-Cell-Lines with specific gene over-expression or knock-down are very helpful in gene function analysis, target discovery, target validation, assay development, and compound screening. However, generation of stable cell lines is a time-consuming and expensive process.

Creative Biogene has established a team of experts for stable cell line generation. We have successfully generated hundreds of stable cell lines including: Gene Overexpression Cell Lines Gene Knockout Cell Lines Gene Knockdown Cell Lines

These cell products are in well preparation to meet the requirements of our customers. Besides, we can also provide PanoplyTM cell products to satisfy your research needs.

Creative Biogene’s cell lines are generated by plasmid transfection/lentivirus transduction, selection of stable cells, colony picking, expression and functional screening, and final validation of construct expression by real-time qRT-PCR or Western blot analysis.

Stable Cell Line Generation Protocol: • Generate a kill curve to determine the optimal selection antibiotic concentration • Transfect cells with desired plasmid construct(s) • Select and expand stable polyclonal colonies • Identify single clones by limited dilution and expansion • Transfer clones and assess expression • Expand and freeze down high expressing clones

Advantages of Our Transfected Stable Cell Lines: • Experienced: Customized cell generation project was managed by experienced scientists throughout the process, from design to delivery. • Cost effective: Creative Biogene saves your money with competitive prices and meets all your needs. • Reliable cell products:Creative Biogene can save your precious time and speed up your research by providing you with reliable cell products. • Broad cell types:Creative Biogene provide different cell types, including HEK293, HeLa, CHO, U2OS etc. • Convenient: Easy online inquiry and ordering. • Secure shipping & Rapid delivery

Glucose Oxidase

The [glucose oxidase]http://www.creative-enzymes.com/product/Native-Aspergillus-Sp-Glucose-Oxidase_789.html enzyme (GOx) also known as notatin (EC number 1.1.3.4) is an oxido-reductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. This enzyme is produced by certain species of fungi and insects and displays antibacterial activity when oxygen and glucose are present.

alcohol oxidase

In enzymology, an [alcohol oxidase]http://www.creative-enzymes.com/product/Native-Candida-Sp-Alcohol-Oxidase_719.html (EC 1.1.3.13) is an enzyme that catalyzes the chemical reaction a primary alcohol + O2 ↔ an aldehyde + H2O2. Thus, the two substrates of this enzyme are primary alcohol and O2, whereas its two products are aldehyde and H2O2. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor.

Pyruvate Decarboxylase

[Pyruvate decarboxylase]http://www.creative-enzymes.com/common/Lyases-PDC_76_1367.html (PDC) is a homotetrameric enzyme that catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide in the cytoplasm. Pyruvate decarboxylase depends on cofactors thiamine pyrophosphate (TPP) and magnesium. PDC contains a β-α-β structure, yielding parallel β-sheets.

Pancreatin

[Pancreatin]http://www.creative-enzymes.com/common/Hydrolases-Pancreatin_75_1106.html is a mixture of several digestive enzymes produced by the exocrine cells of the pancreas. It is composed of amylase, lipase and protease. This mixture is used to treat conditions in which pancreatic secretions are deficient, such as surgical pancreatectomy, pancreatitis and cystic fibrosis. It has been claimed to help with food allergies, celiac disease, autoimmune disease, cancer and weight loss. Pancreatin is sometimes called "pancreatic acid", although it is neither a single chemical substance nor an acid.

Bovine Cathepsin B

[Bovine Cathepsin B]http://www.creative-enzymes.com/common/Hydrolases-Cathepsin-B_75_2244.html has been found to cleave procaspase 1 and procaspase 11 and to induce apoptosis in digitonin-permeabilized cells. Translocation of cathepsin B from the cytoplasm to the nucleus contributes to bile salt induced apoptosis of rat hepatocytes. Levels of cathepsin B in PC12 cells significantly decrease 12 to 24 hours after apoptosis is induced.

itraq proteomics

Creative Proteomics offers iTRAQ protein quantification service suited for unbiased untargeted biomarker discovery. Relative quantification of proteins for biomarker discovery in complex mixtures by mass spectrometry can easily and quickly be achieved using iTRAQ technology. iTRAQ is ideally suited for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and provides relative quantitation.

Protein quantification through incorporation of stable isotopes has become a central technology in modern proteomics research. Isobaric tags for relative and absolute quantitation (iTRAQ) is used in proteomics to study quantitative changes in the proteome.

iTRAQ-based quantification facilitates the comparative analysis of peptide and proteins in a variety of settings including comparison of normal, disease or drug treated states.

The iTRAQ technology utilizes isobaric reagents to label the primary amines of peptides and proteins. The iTRAQ reagents usually consist of an N-methyl piperazine reporter group, a balance group, and an N-hydroxy succinimide ester group that is reactive with the primary amines of peptides. The balance groups present in each of the iTRAQ reagents function to make the labeled peptides from each sample isobaric and the quantification is facilitated through analysis of reporter groups that are generated upon fragmentation in the mass spectrometer. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS).

The iTRAQ workflow contains several steps. Samples to be quantified are prepared under various treatment conditions followed by cell lysis to extract proteins. After using a standard protein assay to estimate the protein concentration of each sample, proteins are digested using an enzyme, such as trypsin, to generate proteolytic peptides. Each peptide digest is labeled with a different iTRAQ reagent and then the labeled digests are combined into one sample mixture. The combined peptide mixture is analyzed by LC-MS/MS for both identification and quantification.

A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.

More information: contact@creative-proteomics.com Tel:5166698109 Web: http://www.creative-proteomics.com/services/itraq-based-proteomics-analysis.htm

determination of amino acid composition

As the basic brick for peptides and proteins , the amino acids are quite important because of the significance of protein/peptide physiological functions. Meanwhile the amino acid composition and levels are essential for nutrition and status of body health. For amino acid metabolomics, there are more than 300 kinds of amino acids and ~20 amino acids can be founded in natural proteins.

Currently there are 3 ways for amino acid analysis: HPLC, automatic amino acid analyzer and LC-MS/MS. In the most experiments, the analytical procedures usually include hydrolysis of protein/peptides in the samples, derivatization of free amino acids pre/post column, separation and quantitation of the amino acids. Not only the 20 natural amino acids, but also all the known amino acids which may exist in the biological samples, such as γ－aminobutyric acid (GABA), L-Ornithine, can be identified and quantified with proper internal standards and calibration curves.

http://www.creative-proteomics.com/services/determination-of-amino-acid-composition.htm