Combined bacterial and fungal targeted amplicon sequencing of respiratory samples: Does the DNA extraction method matter?

High-throughput sequencing techniques were used to analyze the diversity of respiratory microbiota in health and disease. Despite the extensive data available on the respiratory tract bacterial microbiota, the remnants of the less studied fungal components. This is partly due to the technical problems associated with fungal metagenomics analysis. In this study, we compared two protocols of DNA extraction and amplification of two targets for the combined fungal bacteria and fungi that are targeted amplicon sequencing analysis of sputum microbiota.Six breathing, selected randomly from a sample routine Mondor Hospital (Créteil, France) and treated anonymously, were tested after a routine culture of bacteria and fungi.

Two of spiked Fumigati Aspergillus and Aspergillus Nigri (105 conidia / ml). After the mechanical lysis, DNA was extracted using the automatic extraction of QIAsymphony® (AQE) or users PowerSoil® MoBio extraction (MPE). DNA yield and purity compared. DNA extracted from sputum spiked into the target (i) real-time PCR for detection of Aspergillus DNA and (ii) a combination of primary targeting barcode metagenomic analysis for ITS1 and ITS2 fungi, and bacteria V1-V2 and V3-V4 region of the 16S. amplicon libraries were prepared using reagents MiSeq V3 kit on the Illumina platform. Data were analyzed using software PyroMIC © and Shaman, and compared with culture results.AQE extraction provides a higher yield of the (ratio AQE / MPE DNA = 4.5 [1.3 to 11]) DNA in a shorter period of time.

Results of Aspergillus DNA is detected by qPCR were similar to sputum spiked regardless of the extraction protocol. extraction quite an impact on biodiversity or the relative abundance of bacterial communities using targeted amplicon sequencing (2/43 taxa impact). For fungi, the relative abundance of major taxa 4/11 affected and AQE results were closer to the culture results. V1-V2 or V3-V4 and ITS1 or ITS2 target is judged diversity major taxa of bacteria and fungi, but ITS2 and V3-V4 detected smaller taxa.Our results show the importance of joint extraction of DNA for bacteria and fungi targeted metagenomics respiratory samples. Extraction protocol may affect the results of the DNA and the relative abundance of a few bacteria, but more fungal taxa. For the analysis of fungi, ITS2 allow detection of a large number of taxa small compared with ITS1.

ACTG1 human mutation associated with high frequency hearing loss, and patients with mutations in this gene are good candidates for the electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing performed on a 7048 Japanese probands related hearing loss. Among the 1,336 patients with autosomal dominant hearing loss, we identified 15 probands (1.1%) with 13 ACTG1 potentially pathogenic variant. Six new variant and seven were reported previously.

We collect and analyze detailed clinical features of these patients. The average level of development of pure-tone hearing worsening of the average for the four frequency is 1.7 dB / year of age 0-50 years, and all individuals over 60 years old has a severe hearing loss. To better understand the mechanisms underlying causes of disease, the intracellular localization of the wild-type and mutant gamma-actins checked using NIH / 3T3 fibroblast cell line

AHR Western Blot kit (AWBK32243)
AWBK32243 10 reactions
EUR 776.4
Description:
  • Description of target:
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ENG Western Blot kit (AWBK33068)
AWBK33068 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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ID4 Western Blot kit (AWBK38058)
AWBK38058 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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EED Western Blot kit (AWBK38384)
AWBK38384 10 reactions
EUR 776.4
Description:
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SSB Western Blot kit (AWBK40462)
AWBK40462 10 reactions
EUR 776.4
Description:
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OTC Western Blot kit (AWBK41766)
AWBK41766 10 reactions
EUR 776.4
Description:
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FST Western Blot kit (AWBK42287)
AWBK42287 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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TMB Substrate, for Western Blot
F100029 1000 ml
EUR 992.4
Description: TMB Substrate, for Western Blot by Cygnus Technologies is available in Europe via Gentaur.

TMB Substrate, for Western Blot
F129-100 100 ml
EUR 298.8
Description: TMB Substrate, for Western Blot by Cygnus Technologies is available in Europe via Gentaur.

ERG Western Blot kit (AWBK100625)
AWBK100625 10 reactions
EUR 776.4
Description:
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GLI2 Western Blot kit (AWBK31885)
AWBK31885 10 reactions
EUR 776.4
Description:
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PAX4 Western Blot kit (AWBK32064)
AWBK32064 10 reactions
EUR 776.4
Description:
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GLI1 Western Blot kit (AWBK32368)
AWBK32368 10 reactions
EUR 776.4
Description:
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PAX7 Western Blot kit (AWBK32393)
AWBK32393 10 reactions
EUR 776.4
Description:
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LHX6 Western Blot kit (AWBK32553)
AWBK32553 10 reactions
EUR 776.4
Description:
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PAX7 Western Blot kit (AWBK32742)
AWBK32742 10 reactions
EUR 776.4
Description:
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SOX5 Western Blot kit (AWBK33323)
AWBK33323 10 reactions
EUR 776.4
Description:
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A1BG Western Blot kit (AWBK33810)
AWBK33810 10 reactions
EUR 776.4
Description:
  • Description of target:
  • Species reactivity: Human
  • Application:
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NPM1 Western Blot kit (AWBK34094)
AWBK34094 10 reactions
EUR 776.4
Description:
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LIG4 Western Blot kit (AWBK34122)
AWBK34122 10 reactions
EUR 776.4
Description:
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RNF2 Western Blot kit (AWBK34290)
AWBK34290 10 reactions
EUR 776.4
Description:
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ILF2 Western Blot kit (AWBK35731)
AWBK35731 10 reactions
EUR 776.4
Description:
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SPP1 Western Blot kit (AWBK36677)
AWBK36677 10 reactions
EUR 776.4
Description:
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ZBP1 Western Blot kit (AWBK36711)
AWBK36711 10 reactions
EUR 776.4
Description:
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G3BP Western Blot kit (AWBK37713)
AWBK37713 10 reactions
EUR 776.4
Description:
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BRD7 Western Blot kit (AWBK39018)
AWBK39018 10 reactions
EUR 776.4
Description:
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MSI2 Western Blot kit (AWBK41112)
AWBK41112 10 reactions
EUR 776.4
Description:
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TPM1 Western Blot kit (AWBK41392)
AWBK41392 10 reactions
EUR 776.4
Description:
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TSHR Western Blot kit (AWBK41870)
AWBK41870 10 reactions
EUR 776.4
Description:
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ASPN Western Blot kit (AWBK42487)
AWBK42487 10 reactions
EUR 776.4
Description:
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RORA Western Blot kit (AWBK45608)
AWBK45608 10 reactions
EUR 776.4
Description:
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PDCD4 Western Blot kit (AWBK09045)
AWBK09045 10 reactions
EUR 776.4
Description:
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ETS1 Western Blot kit (AWBK100604)
AWBK100604 10 reactions
EUR 776.4
Description:
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GABRP Western Blot kit (AWBK13034)
AWBK13034 10 reactions
EUR 776.4
Description:
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HTR1A Western Blot kit (AWBK13041)
AWBK13041 10 reactions
EUR 776.4
Description:
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RAB14 Western Blot kit (AWBK13107)
AWBK13107 10 reactions
EUR 776.4
Description:
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TNRC4 Western Blot kit (AWBK31525)
AWBK31525 10 reactions
EUR 776.4
Description:
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ESRRG Western Blot kit (AWBK31655)
AWBK31655 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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HIPK2 Western Blot kit (AWBK32586)
AWBK32586 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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ZFP67 Western Blot kit (AWBK32749)
AWBK32749 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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OLIG2 Western Blot kit (AWBK32753)
AWBK32753 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SOX10 Western Blot kit (AWBK33326)
AWBK33326 10 reactions
EUR 776.4
Description:
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RPS16 Western Blot kit (AWBK33535)
AWBK33535 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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ATP7A Western Blot kit (AWBK33797)
AWBK33797 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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ZFP36 Western Blot kit (AWBK34385)
AWBK34385 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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ARIH2 Western Blot kit (AWBK34418)
AWBK34418 10 reactions
EUR 776.4
Description:
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KEAP1 Western Blot kit (AWBK34728)
AWBK34728 10 reactions
EUR 776.4
Description:
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CLIC1 Western Blot kit (AWBK35045)
AWBK35045 10 reactions
EUR 776.4
Description:
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KCNN2 Western Blot kit (AWBK35094)
AWBK35094 10 reactions
EUR 776.4
Description:
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CLCN3 Western Blot kit (AWBK35504)
AWBK35504 10 reactions
EUR 776.4
Description:
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P2RX7 Western Blot kit (AWBK35518)
AWBK35518 10 reactions
EUR 776.4
Description:
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TRPM3 Western Blot kit (AWBK35609)
AWBK35609 10 reactions
EUR 776.4
Description:
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PITX2 Western Blot kit (AWBK35634)
AWBK35634 10 reactions
EUR 776.4
Description:
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ZFP42 Western Blot kit (AWBK36244)
AWBK36244 10 reactions
EUR 776.4
Description:
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ANXA1 Western Blot kit (AWBK36568)
AWBK36568 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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RUNX2 Western Blot kit (AWBK36678)
AWBK36678 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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Bapx1 Western Blot kit (AWBK36826)
AWBK36826 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SATB1 Western Blot kit (AWBK38208)
AWBK38208 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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JMJD3 Western Blot kit (AWBK40102)
AWBK40102 10 reactions
EUR 776.4
Description:
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STAU1 Western Blot kit (AWBK40537)
AWBK40537 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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PCBP1 Western Blot kit (AWBK40630)
AWBK40630 10 reactions
EUR 776.4
Description:
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WNT2B Western Blot kit (AWBK41254)
AWBK41254 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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GNB1L Western Blot kit (AWBK41328)
AWBK41328 10 reactions
EUR 776.4
Description:
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STK11 Western Blot kit (AWBK41743)
AWBK41743 10 reactions
EUR 776.4
Description:
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KRT13 Western Blot kit (AWBK42031)
AWBK42031 10 reactions
EUR 776.4
Description:
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IDH3A Western Blot kit (AWBK42237)
AWBK42237 10 reactions
EUR 776.4
Description:
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PBEF1 Western Blot kit (AWBK42255)
AWBK42255 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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RSAD2 Western Blot kit (AWBK42696)
AWBK42696 10 reactions
EUR 776.4
Description:
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PANX2 Western Blot kit (AWBK42778)
AWBK42778 10 reactions
EUR 776.4
Description:
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PANX1 Western Blot kit (AWBK42783)
AWBK42783 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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TGFBI Western Blot kit (AWBK44268)
AWBK44268 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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PTPN2 Western Blot kit (AWBK45362)
AWBK45362 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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TWIST1 Western Blot kit (AWBK37997)
AWBK37997 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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TARDBP Western Blot kit (AWBK38941)
AWBK38941 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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TARDBP Western Blot kit (AWBK38942)
AWBK38942 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SNRP70 Western Blot kit (AWBK40439)
AWBK40439 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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HNRPLL Western Blot kit (AWBK41102)
AWBK41102 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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HNRPA3 Western Blot kit (AWBK41195)
AWBK41195 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SLC1A5 Western Blot kit (AWBK42247)
AWBK42247 10 reactions
EUR 776.4
Description:
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ONECUT2 Western Blot kit (AWBK35750)
AWBK35750 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SLC12A1 Western Blot kit (AWBK41388)
AWBK41388 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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CEACAM6 Western Blot kit (AWBK41504)
AWBK41504 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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SLC22A1 Western Blot kit (AWBK41516)
AWBK41516 10 reactions
EUR 776.4
Description:
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  • Species reactivity:
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RHOBTB1 Western Blot kit (AWBK41883)
AWBK41883 10 reactions
EUR 776.4
Description:
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SLC22A7 Western Blot kit (AWBK42306)
AWBK42306 10 reactions
EUR 776.4
Description:
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Interferon alpha Western Blot Antibody
20-abx137014
  • EUR 427.20
  • EUR 627.60
  • 0.5 mg
  • 1 mg

Colorimetric Western Blot Kit (OKRA00014)
OKRA00014 1 kit
EUR 730.8
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00015)
OKRA00015 1 each
EUR 730.8
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00016)
OKRA00016 1 kit
EUR 730.8
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Colorimetric Western Blot Kit (OKRA00017)
OKRA00017 1 kit
EUR 730.8
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

cyclin G2 Western Blot kit (AWBK03032)
AWBK03032 10 reactions
EUR 776.4
Description:
  • Description of target:
  • Species reactivity:
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ChonBlock Western Blot Buffer 1 (PBS)
9069P 10 ml
EUR 232.26
Description: ChonBlock Western Blot Buffer 1 (PBS)

ChonBlock Western Blot Buffer 1 (TBS)
9069T 10 ml
EUR 232.26
Description: ChonBlock Western Blot Buffer 1 (TBS)

Pure-IP Western Blot Detection Kit
PRB-5002 20 blots
EUR 470.4
Description: Western blot detection of IP proteins can exhibit high background due to the use of the same primary antibody in the IP and the Western blot. The Pure-IP Western Blot Detection Kit eliminates this background problem by using a proprietary HRP Conjugate for detection of the primary antibody.

Chemiluminescent Western Blot Kit (OKRA00001)
OKRA00001 1 kit
EUR 684
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00002)
OKRA00002 1 kit
EUR 684
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00003)
OKRA00003 1 kit
EUR 684
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Chemiluminescent Western Blot Kit (OKRA00004)
OKRA00004 1 kit
EUR 684
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

T-Pro Western Blot Stripping Reagent
JB11-K002 500ml/BT
EUR 213.6

AgileBlotter? Automated Western Blot Processor
ACT-ABH-40 each
EUR 20326.8

Nitrated Mol Wt Std for Western blot
NITT12-C 100 ul
EUR 343.2

Western Blot Primary Antibody Diluent Buffer
abx090689-100ml 100 ml
EUR 218.4

Attoglow Western Blot Analysis Kit:Blocking Agent
K3171250-3 40 g
EUR 68

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-10 20mL (sufficient reagents for 4000 cm2 of membrane)
EUR 200.4

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-25 50mL (sufficient reagents for 10000 cm2 of membrane)
EUR 376.8

Hypersensitive Western Blot Chemiluminescent Substrate
CR0001-5 10mL (sufficient reagents for 2000 cm2 of membrane)
EUR 139.2

Recombinant Human MutY Protein for Western blot
MUTY11-C 100 ul
EUR 343.2

ChonBlock Western Blot Buffer 2 for Goat (PBS)
90691P 10 ml
EUR 145.26
Description: ChonBlock Western Blot Buffer 2 for Goat (PBS)

ChonBlock Western Blot Buffer 2 for Goat (TBS)
90691T 10 ml
EUR 145.26
Description: ChonBlock Western Blot Buffer 2 for Goat (TBS)

West-Ez Blocking Buffer, 1 % Casein for Western Blot
W3720-050 500ml
EUR 144

West-Ez Blocking Buffer, 1 % Casein for Western Blot
W3720-100 2x500ml
EUR 176.4

Poly-ADP-ribosylated proteins for Western blot
ADPR11-C 100 ul
EUR 343.2

Rat Ceruloplasmin protein control for western blot
CP17-C 100 ul
EUR 343.2

Attoglow Western Blot Analysis Kit:Binding Buffer 20x
K3171250-2 120 ml
EUR 107

SuperBrite? ECL Western Blot Substrate/Detection Kit
K824-50 each
EUR 229.2

ChonBlock Western Blot Buffer 2 for Rabbit (PBS)
90692P 10 ml
EUR 145.26
Description: ChonBlock Western Blot Buffer 2 for Rabbit (PBS)

ChonBlock Western Blot Buffer 2 for Rabbit (TBS)
90692T 10 ml
EUR 145.26
Description: ChonBlock Western Blot Buffer 2 for Rabbit (TBS)

Bovine DNAse 1 Protein Western blot +ve control
DNASE11-C 100 ul
EUR 343.2

Mouse Leptin protein Western blot +ve control # 1
LEP11-C 100 ul
EUR 343.2

Human Leptin protein Western blot +ve control # 2
LEP12-C 100 ul
EUR 343.2

Mouse Ceruloplasmin protein control for western blot
CP16-C 100 ul
EUR 343.2

Western blot recycling stripping buffer, 50 ml (10X)
90101 50 ml
EUR 285.6

Box Western Blot Polystyrene Transparent 95x30x16mm X5
DD62676 PK5
EUR 99.6

Attoglow Western Blot Analysis Kit: Millennium Enhancer
K3171250-1 100 ml
EUR 130

Purified Human C1q protein Western blot +ve control
C1Q11-C 100 ul
EUR 343.2

HRP Western Blot Anti-IgG Antibody Kit (OKRA00006)
OKRA00006 100 ug
EUR 246
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Human C1 Esterase Inhibitor western blot +ve control
C1E11-C 100 ul
EUR 343.2

WesternOne? Marker Pen, HRP for Western blot chemiluminescent detection
ACT-WP each
EUR 172.8

Human Liver Ferritin protein Western blot +ve control
FERT12-C 100 ul
EUR 343.2

NuGlo (luminol) ECL Substrate Kit for Western blot, 250 mls
80215-250 1 kit
EUR 196.8

Beta-Galactosidase (E. Coli) Protein for Western Blot
BGAL11-C 100 ul
EUR 343.2

Human Spleen Ferritin protein Western blot +ve control
FERT11-C 100 ul
EUR 343.2

Horse Spleen Ferritin protein Western blot +ve control
FERT13-C 100 ul
EUR 343.2

Chicken Serum Albumin protein control for Western blot
ALBC15-C 100 ul
EUR 343.2

Purified Cholera Toxin protein control for Western Blot
CTOX11-C 100 ul
EUR 343.2

Human Pancreatic Amylase protein control for Western blot
PAMY13-C 100 ul
EUR 343.2

Western Blot Box - 6 x 4 x 2in.; 15.2 x 10.2 x 5.1cm
B1200-19 5/pack
EUR 108.6
Description: Western Blot Boxes

Camelpox virus H3L/p35 protein control for western blot
CPOX11-C 100 ul
EUR 343.2

Human Adipsin/Factor D protein Western blot +ve control
ADN12-C 100 ul
EUR 343.2

Zika Virus prM Protein (African) control for Western blot
ZPRM11-C 100 ul
EUR 343.2

Recombinant Beta Lactamase protein control for Western blot
BLAC11-C 100 ul
EUR 343.2

Human Liver Cathepsin B protein control for Western blot
CTHB11-C 100 ul
EUR 343.2

Human Liver Cathepsin D protein control for Western blot
CTHD11-C 100 ul
EUR 343.2

Human Liver Cathepsin G protein control for Western blot
CTHG11-C 100 ul
EUR 343.2

Human Liver Cathepsin H protein control for Western blot
CTHH11-C 100 ul
EUR 343.2

Human Liver Cathepsin L protein control for Western blot
CTHL11-C 100 ul
EUR 343.2

HRP Western Blot Anti-Rabbit IgG Antibody Kit (OKRA00007)
OKRA00007 100 ug
EUR 246
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

NuGlo (luminol) ECL Substrate Kit for Western blot, 50 mls
80215-250-S 1 kit
EUR 169.2

Recombinant human BAFF-R protein control for Western blot
BAFFR11-C 100 ul
EUR 343.2

Purified rat serum albumin protein control for Western blot
ALBR12-C 100 ul
EUR 343.2

Purified Ram Cyclooxygenase (Cox-1) Western blot +ve control
COX11-C 100 ul
EUR 343.2

Purified Ram Cyclooxygenase (Cox-2) Western blot +ve control
COX21-C 100 ul
EUR 343.2

Biotin Western Blot Anti-IgG Antibody MaxTag Kit (OKRA00023)
OKRA00023 1 ml
EUR 339.6
Description: Description of target: ;Species reactivity: ;Application: ;Assay info: ;Sensitivity:

Purified Mouse serum albumin protein control for Western blot
ALB11-C 100 ul
EUR 343.2

Purified Human serum albumin protein control for Western blot
ALBH13-C 100 ul
EUR 343.2

Purified Sheep serum albumin protein control for Western blot
ALBSH20-C 100 ul
EUR 343.2

Western Blot Box - 6 x 4 x 1 1/4in.; 15.2 x 10.2 x 3.2cm
B1200-17 5/pack
EUR 107.63
Description: Western Blot Boxes

Western blot Kit for Rat Primary Antibodies, Chemilum. Substrate
80203-Rt 1 kit
EUR 781.2

Western blot Kit for Pig Primary Antibodies; Chemilum. Substrate
80209-Pg 1 kit
EUR 781.2

Mouse NOS-II/i-NOS Protein Western Blot +ve Control # 1
iNOS-C 100 ul
EUR 343.2

Western blot Kit for Goat Primary Antibodies, Chemilum. Substrate
80201-Gt 1 kit
EUR 781.2

Human recombinant, purified BMP2 protein Western blot +ve control
BMP21-C 100 ul
EUR 343.2

Human recombinant, purified BMP3 protein Western blot +ve control
BMP31-C 100 ul
EUR 343.2

Human recombinant, purified BMP4 protein Western blot +ve control
BMP41-C 100 ul
EUR 343.2

Human recombinant, purified BMP5 protein Western blot +ve control
BMP51-C 100 ul
EUR 343.2

Human recombinant, purified BMP6 protein Western blot +ve control
BMP61-C 100 ul
EUR 343.2
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