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A New C-Terminal GST Vector for Protein Production in S. pombe
...the pESP-3 vector, aC-terminal fusion with the GST peptide (~27 kDa) results, which facilitatesone-step purification of the GST fusion protein. 1 In order to provideconvenient removal of the GST tag after purification, a sequence coding for therecognition and cleavage sites for enterokinase and thrombin hav...Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
...ive mass spectrometric techniques provide valuable peptide sequence information from very limited amo...ghly degenerate primers from these partial peptide sequences, but our initial attempts at PCR-cloning were unsuccessful. The peptide sequences ...Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived Tissue
...tecting the rare message for the gastrin-releasing peptide (GRP) receptorexpressed by a previously resected g...tility and to induce the secretion ofother enteric peptide hormones. 1-3 However, GRP receptors are notexpressed by mucosal epithelial cells lining the normal...Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
...y of cloning andexpression vectors. Since the FLAG peptide (DYKDDDDK) is a commonly usedepitope tag, Stratagene developed the TagDetect FLAG Protein Dipstick. It can beused to monitor expression of FLAG-tagged proteins in either prokaryotic oreukaryotic cells, to follow FLAG-tagged proteins during purifica- ...Epitope-Tagging Vectors for Functional Analysis in Yeast
...tagging is a convenient technique in which a known peptide epitopethat is recognized by an antibody is fused to the target protein of interest.With this technique, a tag-specific antibody can be used to detect the fusionprotein within a cell, eliminating the need to make specific antibodies to eachnew protei...New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
...ocedure based on the selective affinity of the GST peptide forglutathione immobilized on resin. Following the single-step purificationprocedure, the GST affinity tag may be removed by proteolytic cleavage with EKto yield polypeptides with native amino acid sequence. Cleavage with thrombinyields the recombina...High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
...y contribute to background. The calmodulin-binding peptide (CBP) purificationtag can be removed from the reco...of theprotein coding sequence of interest to short peptide sequences, or largerproteins, enabling the affinity purification of the fusion protein from crudepre...Sex Steroid Regulation of Vasopressin Levels Secreted by hNT Neurons*
...avoidance behavior and temperatureregulation. 4 A peptide hormone of interest, vasopressin, orantidiuretic hormone (ADH), is made in specialized neurons found in thehypothalamus and then transported to the pituitary gland. In neurons, both ADHcontent and ADH mRNA levels show a marked decline in senescence. ...Yeast Protein Production System Features High Yields and One-Step Purification
...rombin results in a fusion protein having the FLAG peptide (DYKDDDDK) 5 at its N terminus; antibodies against the FLAG peptide are available from Stratagene. Establishing the Expression Strain The gene of interest is ...Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
...sed in frame, downstream of the calmodulin-binding peptide (CBP) purification tag. The resulting fusi...ns and proteolytic removal of the affinity peptide tags, recombinant EK reactions contain a mixture of the protein of interest, the free affini...Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
... sequence is locatedbetween the calmodulin-binding peptide (CBP) purification tag and the N terminusof the expressed protein. Because EK cleaves at the C terminus of itsrecognition target, which is also the N terminus of the inserted polypeptidesequence, cleavage of fusion proteins produced in the pCAL-n-EK ...Highly Active and Removable AffinityTagged Lambda Phosphatase
... a fusion to an affinity tag, thecalmodulinbinding peptide (CBP). 5 The tag simplifies purification ofthe enzyme and allows the enzyme to be removed after dephosphorylation of thetarget protein. As the target protein may be a kinase, removal of thephosphatase is not only convenient but also essential for th...TKB1 Cells Identify Receptor Tyrosine Kinase Interacting Proteins
...pecific manner and,therefore, bind to short linear peptide sequences. 1,2,3 Extensive mutagenesis of known autophosphorylation sites in the RTKNeu/ErbB-2 has revealed that four (Y1144, Y1201, Y1227, Y1253) of the fiveautophosphorylation sites are capable of mediating transformation from thereceptor. 4 Sub...Mammalian and Bacterial Expression in One Vector
...e their target geneto the small calmodulin binding peptide (CBP) affinity tag located downstream ofthe cloning site. The 4-kDa CBP fusion tag, expressed on the C-terminus of theprotein, can then be used for easy detection and purification of the fusionprotein using Stratagenes Affinity CBP fusion detectio...Sample Preparation for Microinjection
...eins or antibodies should be used. For peptide antibodies raised in rabbits, affinity purification is recommended. The concentration of the material injected should be 10 to 20 times higher than that required for the optimum in vitro activity, because the sampl......f tryptic digest of oxidized CaM revealed 13 peptide fragments that were identified in a single Data De... corresponding to an oxidized form of T 12 . This peptide is of particular interest because it contains two possible oxidation sites, Met 109 and...Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
...ein is enzymatically digested, the resulting peptide fragments are separated using HPLC and the p...a glycosidase to cleave the bond between the peptide and the oligosaccharide. The difference in mass following cleavage is used to infer the carboh...... d 8 ratio (bottom right) of the ICAT-labeled peptide TGGVLGIKIGWVC*IPK using XPRESS. Note that in addition to the expected yand b-ions labeled with blue and red in the spectrum, the spectrum also shows two diagnostic ICAT ions at m/z 288 and 485. There were a total of sev...Enhancing Ruggedness and Full-Scan MS Sensitivity Using Ion Sweep Technology
... In assays such as metabolite identification or peptide mapping, the full MS scan plays a pivotal ro...ne noise. Furthermore, metabolism and peptide samples inherently have matrices such as plasma or serum. Therefore, any analytical method use......uniquely identified by database searching of peptide fragmentation spectra. (3) The MS/MS spectr...th of information enabling the assignment of peptide sequences and identification of modified residues. We present a simple strategy enabling an un...A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
...is also shown by immunofluorescencein Figure 2a. A peptide competition control experimentis included (Figure 2b). To gain new insights into apoptotic pathways particularlyin situ Roche Applied Science now offers the anti-PARP Caspase Cleavage Product-Fluorescein antibod...Setting up a kinase profiler with IMAP
...on antibody recognition. Thus virtually any peptide substrate can be easily configured to run i... 50 in the assay was 0.01 U/mL, obtained with the peptide 5FAM-LKKLRRRLSDANF-NH 2 (ID G3H4). This peptide was chosen as the AMPK profiling su...Hands-free Tissue Disruption No Homogenization Needed
...nidine, the MELT reagents digest RNases to peptide fragments that cannot reactivate. As a result, MELT enzymes irreversibly destroy RNases and enable the preservation of intact RNA in tissue lysates for up to six days at ambient temperatures (Figure 5). The ability of the MELT...Translate High Yields of Biologically Active Proteins
... by phosphorylation of casein kinase 1 substrate peptide (Upstate). Summary ActivePro is a superior, cell-free in vitro translation kit delivering both high yields and active protein. It is fast, easy to use, and scalableas m...Purification of Insulin on UNO S1 Column, Rev A
Insulin is a peptide hormone which stimulates anabolic reactions for carbohydrates, proteins, and fats resulting in lowered blood glucose. It is used therapeutically for treatment of diabetes mellitus and was the first recombinant protein on the m......ated in our work to date. The IPQSLDSWWTSL peptide containing the dodecameric class I Ld-restricted epitope of the hepatitis B envelope will be used in cytotoxic T cell assays and tetramer analysis. This not only allows the generation of standardized assays, but also provides ...The Bio-Rad Silver Stain, Rev E
...stains. The incorporation of these amino acidsinto peptide chains, as well as cooperative effects of severali...ferences) including IEF, 2-D, gradient and peptide gels, and nucleic acids in denaturing gels Adjunct to Protein Identification and Characteriza...Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A
...tions among other functions. 2DE and MALDI peptide mass fingerprinting data will be presented...ntLite. The search engine used to identify peptide mass fingerprint data was ProteinLynx Global SERVER 2.0 software. Results ......S data. Statistical analysis is carried out on all peptide spots within minutes. <P >The DeCyder MS a...Detect module of DeCyder MS was used for automated peptide detection, charge-state assignments, deconvolution, and quantitation based on MS signal intensities ......n the pituitary and are processed into three major peptide hormone fragments: adrenocorticotrophin (ACTH), the melanocyte stimulating hormones, and β-endorphin. The various melanocyte stimulating hormones are known to activate the MC 4 (melanocortin subtype 4) receptor. The MC 4 receptor has been link......n the pituitary and are processed into three major peptide hormone fragments: adrenocorticotrophin (ACTH), the melanocyte stimulating hormones, and β-endorphin. The various melanocyte stimulating hormones are known to activate the MC 5 (melanocortin subtype 5) receptor. The melanocortin 5 receptor (MC5...2D-LC analysis of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ
... important to confirm that the charge state of the peptide was correct, that the neutral loss ion dominated the MS/MS spectrum (Fig 4), and that the sequence data was of high quality. <P ><P >Database searches were then performed on all MS/MS spectra, and the results were used to confirm th......g Beads Calcitonin-gene related peptide (CGRP) is believed to have an important physiologi... IM184 related peptide (CGRP) <P > Other material required Human recombinant CGRP ...Sensitive identification of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ
... important to confirm that the charge state of the peptide was correct, that the neutral loss dominated the MS/MS spectrum (Fig 3), and that the sequence data was of high quality. <P > <P >Database searches were then performed on all MS/MS spectra. The results were used to ...Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein
...endogenous protein (a fusion protein) or as a free peptide in the cytoplasm. Owing to the high solubility of EGFP when expressed as a free protein, fixation is required to prevent leaching of the protein and retain fluorescence after cryosectioning. Recently, chromosomal expression of EGFP from transgenic re...Efficient Primary Structure Elucidation of Disulfide-bridged Peptides
...e, enabling rapid protein identification either by peptide mass mapping or by determination of partial amino ... was added prior to analysis, resulting in a final peptide concentration of 5 picomoles per microliter. The instrument was tuned for a resolving power of 12'00......d quantitate selected tryptic peptides, where each peptide represents a specific candidate protein biomarker ...roteomics experiments, MRMs were designed based on peptide MS/MS spectra. For the remaining candidates, MRMs were developed either by in silico design, based o...Direct Monitoring of Solid-Phase Peptide Synthesis by MALDI-TOF MS
...n to be a valuable tool in verifying and improving peptide synthesis. Matrix Assisted Laser Desorption Ioniza...MALDI-TOF MS to follow the progress of solid phase peptide synthesis. This work was first presented by A. Falick, G. Tarr and N. Sole at ABRF 96. Key F...Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System
... fluorescent signal will be detected when the IL-8 peptide is present in the media and the bead/antibody/peptide complexes settle to the bottom of the well. To measure cell surface expression of ICAM-1, a dye B-labeled anti-ICAM-1 antibody is added simultaneously with the beads and IL-8 detection antibodies ......ding BFP and GFP linked by a 27 amino acid peptide linker that contains a recognition site fo...ver whole cells to allow protease digestion of the peptide linker. Y-PER lysates (10l) were diluted with 1.2ml Tris/HCl pH 8 and placed in disposable f...Small-Molecule Quantitation Using Automated Single-Shot Averaging MALDI-TOF Mass Spectrometry
...F mass spectrometry for small organic molecule and peptide quantitation has become a viable analytical procedure. Robotic or manual sample preparation combined with automated MALDI-TOF mass spectrometry, using single-shot averaging, is now a viable method for quantitation of small organic molecules and pepti......se MALDI-TOF mass spectrometry can easily generate peptide mass fingerprint profiles and relative quantitatio...tify the ICAT reagent pairs based on a theoretical peptide mass list from known protein digests, which could lead to accurate identification of ICAT reagent pa...... Bands from the 1D Gel were further analyzed using peptide mass fingerprinting analysis on the VOYAGER Workst...the proteins visualized by silver stain. MALDI-TOF peptide mass fingerprinting (PMF) was performed on bands from 1D Gel that did not correspond to the intact m...Cerenkov Counting Performance on the TopCountTM Microplate Scintillation and Luminescence Counter
...ple by the degree of phosphorylation of a specific peptide with [ 32 P]-ATP. The assay was performed essentially as described in TopCount Topics, TCA-018. 4 Serial dilutions of PKC (P-8289, Sigma ) were used to generate several levels of specific kinase activity ranging from 125 to 7.8 units of activity pe...Phosphorescence lifetime measured using the SPEX phosphorimeter
...rbium-complex donor to a fluorescein acceptor. The peptide in the complex absorbs light at 280 nm, the terbiu...With the delay,the fluorescence at 363 nm from the peptide disappears,isolating the Tb phosphorescence at 486 and540 nm. Figure 2 shows fluorescence from a sol...Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers
...f whether it is free in solution, derivatized to a peptide or, while covalently attached to that peptide, als... In the case of a fluorescent derivative of a peptide bound to a GPCR embedded in a cell membrane fragment, little rotation can occur over the fluorescenc...Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.
...tilin MTL-R1A receptor. Motilin is a 22-amino acid peptide secreted from endocrine cells of the small intestine that stimulates gastrointestinal contractility (Tonini et al ., 1996). The receptor for motilin has been cloned recently (Feighner et al ., 1999) and belongs to the GPCR family. Development of mo...Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers
...ent manner, as expected (Figure 2a-2c). (ALLN is a peptide aldehyde that inhibits the proteasomes chymotrypsi...r, E.M. Calpain inhibitor I increases beta-amyloid peptide production by inhibiting the degradation of the substrate of gamma-secretase. Evidence that substrat......ll microplate. The calibrator is a phosphotyrosine peptide that closely mimics the assay response to tyrosine kinase products. This curve illustrates the nanomolar sensitivity of the TKXtra kit. Optimization of β− Insulin receptor kinase ( β -IRk) tyrosine kinase: ......ypically use synthetic chromogenic or flu-orogenic peptide substrates. Enzyme selectivity is obtained by choosing a peptide sequence uniquely recognized by the catalytic site of a particular enzyme. The chromophore or fluoro......mpartments with appropriateregulatory elements and peptide signals 3 . Moreover, the aequorin complex doesnot require light excitation, thus eliminating autofluorescence, photobleachingand biological degradation problems. Its high affinity for Ca ++ (Kd = 10 M) makes aequorin a good sensor in the biological...Calcium Plus Assay Kit, Superior Detection for Specialized Targets
...ay Kit is designed for use with chemokineand small peptide targets, which can beproblematic and produce less than optimalresults with standard assay formats. IMPROVED RESULTS The primary advantage of the Calcium PlusAssay Kit is the improvement in the data quality.When compared with ...FLIPR Calcium 3 Assay Kit Datasheet
...lular calcium fluxes even from chemokine and small peptide targets, as well as transient, endogenous and low-expression receptors that have been difficult to assay with standard methods. Strong signals are attained in cases that may previously have yielded weak signal intensity or no peak at all. Thus, the F...... the samples at a higher flow rate or the use of a peptide trap. The peptide trap has several advantages compared to other loading techniques; peptides can be loaded at a higher...Complex Peptide Mixture Analysis on the Finnigan LTQ Linear Ion Trap Mass Spectrometer
...ols to boost sample throughput, and produce higher peptide coverage with a higher degree of confidence for pr... charged ions. Results The plasma peptide mixture was analyzed under two different LC gradients on the Finnigan LTQ and Finnigan LCQ Deca XP P...Protein Depletion for Plasma and Serum Proteomic Analysis
...tidimensional technologies for protein and peptide separation vary in principle and instrumen... or fractions are digested with trypsin to yield peptide fragments. With or without another separation step, the tryptic peptides are analyzed ...Separation of Peptides by Hydrophilic Interaction Chromatography Using TSKgel Amide-80
... The effect of ACN concentration on peptide retention was also examined. Opposite of R...ility between columns. Ten replicates of a peptide mixture were injected on a new TSKgel Amide-80 column and a TSKgel Amide-80 column that had ...LC-MALDI and MALDI-MS/MS for the Analysis of Complex Peptide Mixtures
...paration) acquisitions for the analysis of complex peptide mixtures. These include (i) concentration and separation of the peptides prior to analysis, (ii) extended time available for MS/MS interrogation, (iii) separation of isobaric peptides resulting in increased sequence coverage. The ability to separate ...Glossary for The BioPharm Guide to Fermentation and Cell Culture
... fusion partner When making a small protein or peptide in E. coli , it is often necessary to produce the protein fused to a larger protein to get high levels of stable expression. The resulting fusion protein must be cleaved (chemically or enzymatically broken) t......ylation Independent Lysosomal Targeting or GILT, a peptide tag replaces a chemical component found on some of the enzyme's carbohydrates. The tag is embedded within the therapeutic enzyme to promote internalization into the cell, targeting the enzyme to the lysosome and prolonging the life of enzymes in the ...Other ContentsmicropylemicrosporemicrosporangiummicrotubulesmicrotubulesmicrotubulesmidbrainmidbrainmigrationmigrationmigrationmigrationmigrationmigrationmigrationmineralsmineralsmineralsmineralsmineralsOther TagsKazakhRaeRae
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