Histones are the first protein element of chromatin and are concerned in just about all DNA-templated processes. Histones are abundantly post-translationally modified by quite a lot of chromatin-modifying equipment. These post-translational modifications (PTMs) are acknowledged by a variety of “reader” proteins, which recruit further proteins to particular places on chromatin and impart exact and highly effective results on gene regulation. Every PTM sometimes exerts a constructive or unfavorable impact on transcription, and up to date research have proven that histone PTMs operate in a combinatorial histone code: that’s, histone PTMs operate together to exert exact DNA-templated regulation. Thus, there’s a must determine and perceive proteoforms, or unambiguously outlined single protein molecules with all mixtures of modifications.
High-down proteomics is at present the one viable strategy for figuring out and quantitating histone proteoforms, and mass spectrometry devices have turn into sufficiently highly effective to carry out these quantitative analyses in a strong and high-throughput style. These latest improvements have enabled new experimental instructions in chromatin analysis however have additionally launched temporal and different constraints. This has led us to develop the protocols described right here, which enhance throughput, cut back pattern necessities, and keep sturdy quantitation. Though initially designed for high-throughput quantitative top-down proteomics, the protocols described listed below are helpful for a variety of chromatin biology purposes.
Beginning with small quantities of cells or tissue, we describe two fundamental protocols for exceptionally fast and environment friendly nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone households. We moreover describe the quantitative top-down proteomic evaluation of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Fundamental Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in tradition/tissues Fundamental Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Assist Protocol: Preparation of intact H3 histone tails by Glu-C digestion
Evaluation of 1508 Plasma Samples by Capillary-Move Knowledge-Impartial Acquisition Profiles Proteomics of Weight Loss and Upkeep
Complete, excessive throughput evaluation of the plasma proteome has the potential to allow holistic evaluation of the well being state of a person. Based mostly on our personal expertise and the analysis of latest large-scale plasma mass spectrometry (MS) primarily based proteomic research, we recognized two excellent challenges: gradual and delicate nano-flow liquid chromatography (LC) and irreproducibility of identification of data-dependent acquisition (DDA). We decided an optimum answer decreasing these limitations with sturdy capillary-flow data-independent acquisition (DIA) MS. This platform can measure 31 plasma proteomes per day.
Utilizing this setup, we acquired a large-scale plasma examine of the eating regimen, weight problems and genes dietary (DiOGenes) comprising 1508 samples. Proving the robustness, the entire acquisition was achieved on a single analytical column. Completely, 565 proteins (459 recognized with two or extra peptide sequences) had been profiled with 74% information set completeness. On common 408 proteins (5246 peptides) had been recognized per acquisition (319 proteins in 90% of all acquisitions). The workflow reproducibility was assessed utilizing 34 high quality management swimming pools acquired at common intervals, leading to 92% information set completeness with CVs for protein measurements of 10.9%.
The profiles of 20 apolipoproteins may very well be profiled revealing distinct adjustments. The load loss and weight upkeep resulted in sustained results on low-grade irritation, in addition to steroid hormone and lipid metabolism, indicating useful results. Comparability to different large-scale plasma weight reduction research demonstrated excessive robustness and high quality of biomarker candidates recognized. Monitoring of nonenzymatic glycation indicated a delayed, slight discount of glycation within the weight upkeep section. Utilizing stable-isotope-references, we might instantly and completely quantify 60 proteins within the DIA. In conclusion, we current herein the primary large-scale plasma DIA examine and one of many largest scientific analysis proteomic research to this point. Utility of this quick and sturdy workflow has nice potential to advance biomarker discovery in plasma.
Comparability of Proteomic Applied sciences for Blood-Based mostly Detection of Colorectal Most cancers
Blood-based protein biomarkers are more and more being explored as supplementary or environment friendly alternate options for population-based screening of colorectal most cancers (CRC). The target of the present examine was to check the diagnostic potential of proteins measured with completely different proteomic applied sciences. The concentrations of protein biomarkers had been measured utilizing proximity extension assays (PEAs), liquid chromatography/a number of response monitoring-mass spectrometry (LC/MRM-MS) and quantibody microarrays (QMAs) in plasma samples of 56 CRC sufferers and 99 individuals freed from neoplasms. In one other strategy, proteins had been measured in serum samples of 30 CRC circumstances and 30 individuals freed from neoplasm utilizing immunome full-length purposeful protein arrays (IpAs).
From all of the measurements, 9, 6, 35 and 14 protein biomarkers overlapped for comparative analysis of (a) PEA and LC/MRM-MS, (b) PEA and QMA, (c) PEA and IpA, and (d) LC/MRM-MS and IpA measurements, respectively. Correlation evaluation was carried out, together with calculation of the world below the curve (AUC) for assessing the diagnostic potential of every biomarker. DeLong’s take a look at was carried out to evaluate the variations in AUC.
 ELISA Kit) Mouse Osteonectin (ON) ELISA Kit |
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| RDR-ON-Mu-48Tests | Reddot Biotech | 48 Tests | EUR 511 |
Mouse Osteonectin (ON) ELISA Kit |
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| RDR-ON-Mu-96Tests | Reddot Biotech | 96 Tests | EUR 709 |
 ELISA Kit) Rat Osteonectin (ON) ELISA Kit |
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| RDR-ON-Ra-48Tests | Reddot Biotech | 48 Tests | EUR 534 |
Rat Osteonectin (ON) ELISA Kit |
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| RDR-ON-Ra-96Tests | Reddot Biotech | 96 Tests | EUR 742 |
 ELISA Kit) Human Osteonectin (ON) ELISA Kit |
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| RD-ON-Hu-48Tests | Reddot Biotech | 48 Tests | EUR 478 |
Human Osteonectin (ON) ELISA Kit |
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| RD-ON-Hu-96Tests | Reddot Biotech | 96 Tests | EUR 662 |
Mouse Osteonectin (ON) ELISA Kit |
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| RD-ON-Mu-48Tests | Reddot Biotech | 48 Tests | EUR 489 |
Mouse Osteonectin (ON) ELISA Kit |
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| RD-ON-Mu-96Tests | Reddot Biotech | 96 Tests | EUR 677 |
Rat Osteonectin (ON) ELISA Kit |
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| RD-ON-Ra-48Tests | Reddot Biotech | 48 Tests | EUR 511 |
Rat Osteonectin (ON) ELISA Kit |
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| RD-ON-Ra-96Tests | Reddot Biotech | 96 Tests | EUR 709 |
Human Osteonectin (ON) ELISA Kit |
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| DLR-ON-Hu-48T | DL Develop | 48T | EUR 479 |
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Description: A sandwich quantitative ELISA assay kit for detection of Human Osteonectin (ON) in samples from serum, plasma, tissue homogenates or other biological fluids. |
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Human Osteonectin (ON) ELISA Kit |
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| DLR-ON-Hu-96T | DL Develop | 96T | EUR 621 |
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Description: A sandwich quantitative ELISA assay kit for detection of Human Osteonectin (ON) in samples from serum, plasma, tissue homogenates or other biological fluids. |
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Mouse Osteonectin (ON) ELISA Kit |
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| DLR-ON-Mu-48T | DL Develop | 48T | EUR 489 |
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Description: A sandwich quantitative ELISA assay kit for detection of Mouse Osteonectin (ON) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. |
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Mouse Osteonectin (ON) ELISA Kit |
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| DLR-ON-Mu-96T | DL Develop | 96T | EUR 635 |
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Description: A sandwich quantitative ELISA assay kit for detection of Mouse Osteonectin (ON) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. |
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Rat Osteonectin (ON) ELISA Kit |
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| DLR-ON-Ra-48T | DL Develop | 48T | EUR 508 |
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Description: A sandwich quantitative ELISA assay kit for detection of Rat Osteonectin (ON) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. |
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Rat Osteonectin (ON) ELISA Kit |
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| DLR-ON-Ra-96T | DL Develop | 96T | EUR 661 |
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Description: A sandwich quantitative ELISA assay kit for detection of Rat Osteonectin (ON) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. |
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 Trypsin, Recombinant, Proteomics grade |
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| P1228-1000 | Biovision | EUR 196 | |
Trypsin, Recombinant, Proteomics grade |
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| P1228-10000 | Biovision | EUR 1224 | |
Trypsin, Recombinant, Proteomics grade |
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| P1228-5000 | Biovision | EUR 615 | |
 Biotin-LC-LC-NHS |
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| 2346-1000 | Biovision | EUR 1675 | |
Biotin-LC-LC-NHS |
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| 2346-250 | Biovision | EUR 588 | |
Biotin-LC-LC-NHS |
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| 2346-50 | Biovision | EUR 196 | |
Biotin-LC-LC-NHS |
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| 2346-5000 | Biovision | Ask for price | |
 HemoVoid™ Kit |
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| HVK-05 | Biotech Support Group | 5 Preps | EUR 400 |
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Description: Hemoglobin Removal Kit |
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HemoVoid™ Kit |
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| HVK-10 | Biotech Support Group | 10 preps | EUR 587 |
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Description: Hemoglobin Removal Kit |
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HemoVoid™ Kit |
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| HVK-100 | Biotech Support Group | 100 preps | EUR 2983 |
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Description: Hemoglobin Removal Kit |
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HemoVoid™ Kit |
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| HVK-50 | Biotech Support Group | 50 preps | EUR 1846 |
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Description: Hemoglobin Removal Kit |
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 HemoVoid Binding Buffer HVBB |
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| HVBB-1000 | Biotech Support Group | 1000 mL | EUR 456 |
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Description: HemoVoid™ Buffer |
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HemoVoid Binding Buffer HVBB |
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| HVBB-500 | Biotech Support Group | 500 mL | EUR 245 |
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Description: HemoVoid™ Buffer |
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 HemoVoid Buffer Kit - 1000 |
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| HVBK-1000 | Biotech Support Group | 1000 mL | EUR 1166 |
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Description: HemoVoid™ Buffer |
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 HemoVoid Buffer Kit - 500 |
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| HVBK-500 | Biotech Support Group | 500 mL | EUR 615 |
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Description: HemoVoid™ Buffer |
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 HemoVoid Elution Buffer HVEB |
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| HVEB-1000 | Biotech Support Group | 1000 mL | EUR 456 |
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Description: HemoVoid™ Buffer |
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HemoVoid Elution Buffer HVEB |
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| HVEB-500 | Biotech Support Group | 500 mL | EUR 245 |
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Description: HemoVoid™ Buffer |
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 HemoVoid Wash Buffer HVWB |
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| HVWB-1000 | Biotech Support Group | 1000 mL | EUR 456 |
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Description: HemoVoid™ Buffer |
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HemoVoid Wash Buffer HVWB |
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| HVWB-500 | Biotech Support Group | 500 mL | EUR 245 |
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Description: HemoVoid™ Buffer |
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 (3 biotin/BSA)) Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (3 biotin/BSA) |
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| 7097-25 | Biovision | EUR 222 | |
Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (3 biotin/BSA) |
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| 7097-5 | Biovision | EUR 115 | |
 (5 biotin/BSA)) Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (5 biotin/BSA) |
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| 7098-25 | Biovision | EUR 233 | |
Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (5 biotin/BSA) |
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| 7098-5 | Biovision | EUR 120 | |
 (12 biotin/BSA)) Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (12 biotin/BSA) |
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| 7099-25 | Biovision | EUR 244 | |
Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) (12 biotin/BSA) |
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| 7099-5 | Biovision | EUR 131 | |
 Micro Bead Sterlizer |
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| B1201-E | BenchMark | 1 PC | EUR 542.7 |
Micro Bead Sterlizer |
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| B1202-E | BenchMark | 1 PC | EUR 84.14 |
 Refill Glass Beads |
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| B1201-BEAD | BenchMark | 1 PC | EUR 117.78 |
 101Bio Protein Solubilization Reagent for MS |
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| P5W11 | 101Bio | - | Ask for price |
 BrightGreen miRNA qPCR MasterMix-No Dye |
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| MasterMix-mS | ABM | 4 x 1.25 ml for 500 reactions | EUR 151 |
 MS-1020 |
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| 1911-1 | Biovision | EUR 164 | |
 MS 275 |
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| abx076834-1mg | Abbexa | 1 mg | EUR 314 |
 Ms-275 |
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| 20-abx182582 | Abbexa |
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MS-1020 |
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| GL5031-1MG | Glentham Life Sciences | 1 mg | EUR 160 |
) LC-Protein Kinase G Substrate; LC-Gsubtide (Biotin) |
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| 20-abx265657 | Abbexa |
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 CD49f LC antibody |
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| 70R-49925 | Fitzgerald | 100 ul | EUR 244 |
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Description: Purified Polyclonal CD49f LC antibody |
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 CD49e LC antibody |
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| 70R-49932 | Fitzgerald | 100 ul | EUR 244 |
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Description: Purified Polyclonal CD49e LC antibody |
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 Sulfo-LC-SPDP |
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| 2325-250 | Biovision | EUR 1567 | |
Sulfo-LC-SPDP |
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| 2325-50 | Biovision | EUR 436 | |
 Biotin-LC-NHS |
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| 2345-1000 | Biovision | EUR 1001 | |
Biotin-LC-NHS |
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| 2345-250 | Biovision | EUR 419 | |
Biotin-LC-NHS |
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| 2345-50 | Biovision | EUR 142 | |
 pPINK- LC Plasmid |
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| PVT4035 | Lifescience Market | 2 ug | EUR 325 |
 Biotin LC hydrazide |
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| HY-101885 | MedChemExpress | 5mg | EUR 119 |
 sulfo-LC-SPDP |
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| ADC-L-133 | Creative Biolabs | unit | Ask for price |
 CD224 LC Antibody |
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| 20-abx121054 | Abbexa |
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 Enteropeptidase LC Antibody |
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| 20-abx121089 | Abbexa |
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 MASP1 LC Antibody |
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| 20-abx121154 | Abbexa |
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 Myeloperoxidase LC Antibody |
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| 20-abx121172 | Abbexa |
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 CD49e LC Antibody |
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| 20-abx009097 | Abbexa |
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 CD49f LC Antibody |
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| 20-abx009102 | Abbexa |
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 Biotin-LC-Kemptide |
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| 5-00800 | CHI Scientific | 4 x 1mg | Ask for price |
-Biotin) sulfo-LC-(+)-Biotin |
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| 20-abx185570 | Abbexa |
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-Biotin hydrazide) LC-(+)-Biotin hydrazide |
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| abx185659-1g | Abbexa | 1 g | EUR 648 |
-Biotin) LC-NHS-(+)-Biotin |
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| 20-abx185861 | Abbexa |
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 Kininogen LC antibody |
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| 23031-100ul | SAB | 100ul | EUR 390 |
 NHS-LC-Biotin |
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| A8004-100 | ApexBio | 100 mg | EUR 119 |
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Description: NHS-LC-biotin (succinimidyl-6-(biotinamindo)hexanoate), also known as NHS-X-biotin is a derivative of D-biotin, a amine-reactive biotinylation agent, that contains a spacer arm off the valeric acid side chain of D-biotin with an NHS ester group at its end. |
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NHS-LC-Biotin |
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| A8004-1000 | ApexBio | 1 g | EUR 548 |
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Description: NHS-LC-biotin (succinimidyl-6-(biotinamindo)hexanoate), also known as NHS-X-biotin is a derivative of D-biotin, a amine-reactive biotinylation agent, that contains a spacer arm off the valeric acid side chain of D-biotin with an NHS ester group at its end. |
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 Iodoacetyl-LC-Biotin |
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| A8009-100 | ApexBio | 100 mg | EUR 264 |
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Description: Iodoacetyl-LC-Biotin (N-iodoacetyl-N-biotinylhexylenediamine), a sulfhydryl-reactive biotinylation agent, is a water-insoluble reagent that requires the dissolution of suitable solvents, including dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). |
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 uv lamp 2x15w, 254/365nm |
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| VL215-LC | Consort | ea | EUR 817 |
 uv lamp 2x6w, 254/365nm |
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| VL6-LC | Consort | ea | EUR 538 |
 darkroom 254/365nm |
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| VLCN15-LC | Consort | ea | EUR 1535 |
 Bangs Lab Bead Solution |
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| SOLN1-1000 | Bangs Laboratories | 1000 ML | EUR 155.06 |
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Description: Bangs Lab Bead Solution is ready-to-use solution and is suitable for dilution and/or storage of plain, dyed, or functionalized polymer microspheres |
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Bangs Lab Bead Solution |
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| SOLN1-2000 | Bangs Laboratories | 2000 ML | EUR 212.7 |
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Description: Bangs Lab Bead Solution is ready-to-use solution and is suitable for dilution and/or storage of plain, dyed, or functionalized polymer microspheres |
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Bangs Lab Bead Solution |
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| SOLN1-500 | Bangs Laboratories | 500 ML | EUR 98.51 |
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Description: Bangs Lab Bead Solution is ready-to-use solution and is suitable for dilution and/or storage of plain, dyed, or functionalized polymer microspheres |
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) ELISA kit for Human MS (Melatonin Sulfate) |
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| E-EL-H1726 | Elabscience Biotech | 1 plate of 96 wells | EUR 534 |
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Description: A competitive ELISA kit for quantitative measurement of Human MS (Melatonin Sulfate) in samples from Serum, Plasma, Cell supernatant |
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) ELISA kit for Human Melatonin sulfate (MS) |
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| KTE61523-48T | Abbkine | 48T | EUR 354 |
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Description: Quantitative sandwich ELISA for measuring Human Melatonin sulfate (MS) in samples from cell culture supernatants, serum, whole blood, plasma and other biological fluids. |
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ELISA kit for Human Melatonin sulfate (MS) |
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| KTE61523-5platesof96wells | Abbkine | 5 plates of 96 wells | EUR 2252 |
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Description: Quantitative sandwich ELISA for measuring Human Melatonin sulfate (MS) in samples from cell culture supernatants, serum, whole blood, plasma and other biological fluids. |
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ELISA kit for Human Melatonin sulfate (MS) |
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| KTE61523-96T | Abbkine | 96T | EUR 572 |
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Description: Quantitative sandwich ELISA for measuring Human Melatonin sulfate (MS) in samples from cell culture supernatants, serum, whole blood, plasma and other biological fluids. |
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 HemoVoid™ - Blood Card Reagent Kit |
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| HVBC-10 | Biotech Support Group | 10 preps | EUR 552 |
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Description: Hemoglobin Removal Kit |
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HemoVoid™ - Blood Card Reagent Kit |
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| HVBC-100 | Biotech Support Group | 100 preps | EUR 2937 |
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Description: Hemoglobin Removal Kit |
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HemoVoid™ - Blood Card Reagent Kit |
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| HVBC-50 | Biotech Support Group | 50 preps | EUR 1770 |
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Description: Hemoglobin Removal Kit |
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 Ab MS GluR1* |
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| P80000-100uL | Pel-Freez | 100uL | Ask for price |
) MS-275 (Entinostat) |
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| E1KS1053 | EnoGene | 25 mg | EUR 247 |
 Methyl-PEG3-MS |
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| abx186609-250mg | Abbexa | 250 mg | EUR 439 |
 MS 245 oxalate |
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| B5540-10 | ApexBio | 10 mg | EUR 292 |
MS 245 oxalate |
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| B5540-5 | ApexBio | 5 mg | EUR 210 |
 Biotin-LC-Protein Kinase G Substrate; Biotin-LC-G-Subtide |
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| 5-00802 | CHI Scientific | 4 x 5mg | Ask for price |
 Complement C1R LC antibody |
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| 70R-49435 | Fitzgerald | 100 ul | EUR 244 |
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Description: Purified Polyclonal Complement C1R LC antibody |
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 Cathepsin D LC antibody |
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| 70R-49618 | Fitzgerald | 100 ul | EUR 244 |
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Description: Purified Polyclonal Cathepsin D LC antibody |
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 Sulfo-NHS-LC-Biotin |
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| 2326-100 | Biovision | EUR 311 | |
Sulfo-NHS-LC-Biotin |
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| 2326-50 | Biovision | EUR 202 | |
Sulfo-NHS-LC-Biotin |
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| 2326-set | Biovision | EUR 213 | |
 Cathepsin C LC Antibody |
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| 20-abx121043 | Abbexa |
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 CD41 LC 1 Antibody |
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| 20-abx121061 | Abbexa |
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 Factor IX LC Antibody |
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| 20-abx121094 | Abbexa |
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 Factor XI LC Antibody |
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| 20-abx121097 | Abbexa |
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 CD49f LC Blocking Peptide |
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| 20-abx062800 | Abbexa |
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 CD49e LC Blocking Peptide |
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| 20-abx062807 | Abbexa |
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 Cathepsin D LC Antibody |
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| 20-abx009376 | Abbexa |
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 Complement C1R LC Antibody |
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| 20-abx009532 | Abbexa |
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 Peptide) LC-Kemptide (Biotin) Peptide |
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| 20-abx265999 | Abbexa |
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 CD224 LC Blocking Peptide |
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| 20-abx161054 | Abbexa |
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 Enteropeptidase LC Blocking Peptide |
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| 20-abx161089 | Abbexa |
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 MASP1 LC Blocking Peptide |
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| 20-abx161154 | Abbexa |
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 Myeloperoxidase LC Blocking Peptide |
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| 20-abx161172 | Abbexa |
|
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 Polyclonal CD49e LC Antibody |
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| APR05533G | Leading Biology | 0.1ml | EUR 484 |
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Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human CD49e LC . This antibody is tested and proven to work in the following applications: |
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Sulfo-NHS-LC-Biotin |
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| A8003-100 | ApexBio | 100 mg | EUR 142 |
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Description: Sulfo-NHS-LC-biotin (Sulfosuccinimidyl 6-(biotinamido) Hexanoate) is an amine-reactive biotinylation agent. |
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Sulfo-NHS-LC-Biotin |
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| A8003-1000 | ApexBio | 1 g | EUR 572 |
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Description: Sulfo-NHS-LC-biotin (Sulfosuccinimidyl 6-(biotinamido) Hexanoate) is an amine-reactive biotinylation agent. |
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 CM Rapid Run Agarose Bead |
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| CMRR-25 | ABTBeads | 25 ml | EUR 102 |
CM Rapid Run Agarose Bead |
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| CMRR-300 | ABTBeads | 300 ml | EUR 492 |
 SP Rapid Run Agarose Bead |
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| SPRR-25 | ABTBeads | 25 ml | EUR 102 |
SP Rapid Run Agarose Bead |
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| SPRR-300 | ABTBeads | 300 ml | EUR 492 |
 DEAE Rapid Run Agarose Bead |
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| DEAERR-25 | ABTBeads | 25 ml | EUR 102 |
DEAE Rapid Run Agarose Bead |
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| DEAERR-300 | ABTBeads | 300 ml | EUR 492 |
Analysis of the 9 biomarkers measured with PEA and LC/MRM-MS displayed correlation coefficients >+0.6, related AUCs and DeLong’s p-values indicating no variations in AUCs for biomarkers like insulin-like development issue binding protein 2 (IGFBP2), matrix metalloproteinase 9 (MMP9) and serum paraoxonase lactonase 3 (PON3). Evaluating six proteins measured with PEA and QMA confirmed good correlation and related diagnostic efficiency for just one protein, development differentiation issue 15 (GDF15).