BirA⋆-protein A fusion protein primarily based BioEnhancer amplifies Westernblot immunosignal
Western blot (protein immunoblot) is a extensively used analytical approach in molecular biology. Using the precise recognizing major antibody, proteins immobilized on numerous matrix are investigated by subsequent visualization steps, for instance, by the HRP conjugated secondary antibody incubation. Strategies to enhance the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a brand new technique to amplify Western blot indicators by establishing a probe with proximal labeling and IgG focusing on skills. The R118G mutation attenuated the biotin-AMP binding affinity of the bacterial biotin ligase BirA⋆, providing a proximity-dependent labeling means, which could possibly be used as a sign amplifier. We constructed a BirA⋆-protein A fusion protein (BioEnhancer) which particularly binds to IgG and provides biotin tags to its proximal amine teams, enhancing the immunosignal of goal proteins. In our experiments, the BioEnhancer system amplified the immunosignal by 10-fold in comparison with the usual Western blot. Moreover, our technique might couple with different sign enhancement strategies to additional improve the Western blot sensitivity. This text is protected by copyright. All rights reserved.
A quantitative westernblot approach utilizing TMB: Comparability with the standard approach
Quite a few molecular organic experiments carried out all through the world require the detection or quantification of a protein of curiosity. Western blotting is among the hottest methods used for this goal and affords quantitative info with assistance from specialised software program. Nonetheless, its dependence on the image that’s captured, and the background and the absence of a typical protocol stop the approach from being fully quantitative.
To beat these obstacles, we current a easy and dependable assay that’s much like the common approach, except the final stage of band visualization and quantification. We suggest that small items of the blot that embody the protein of curiosity could be minimize and dipped in a small quantity of three,3′,5,5′-Tetramethylbenzidine (TMB) resolution, giving a colorimetric sign with linear dependence on the amount of the protein.
The response is stopped with H2 SO4 , and the sign is measured in a plate reader. This modification exhibits excessive linearity with out extra prices and could be utilized for each purified proteins and proteins present in a lysate. The outcomes obtained with our proposed approach had been in contrast with these obtained by the standard technique and proved to be extra dependable.
Description: A sandwich quantitative ELISA assay kit for detection of Human Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Human Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A sandwich quantitative ELISA assay kit for detection of Human Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Mouse Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A sandwich quantitative ELISA assay kit for detection of Mouse Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Mouse Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A sandwich quantitative ELISA assay kit for detection of Mouse Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Rat Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A sandwich quantitative ELISA assay kit for detection of Rat Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Rat Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A sandwich quantitative ELISA assay kit for detection of Rat Kelch Like ECH Associated Protein 1 (KEAP1) in samples from serum, plasma, tissue homogenates or other biological fluids.
Rat Kelch Like ECH Associated Protein 1 (KEAP1) ELISA Kit
Description: A polyclonal antibody against KEAP1. Recognizes KEAP1 from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, IHC;ELISA:1:2000-1:5000, IHC:1:25-1:100
Description: A polyclonal antibody against KEAP1. Recognizes KEAP1 from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: WB, ELISA;WB:1/500-1/2000.ELISA:1/20000
Description: A polyclonal antibody against KEAP1. Recognizes KEAP1 from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, IHC;ELISA:1:2000-1:5000, IHC:1:25-1:100
Description: A polyclonal antibody against KEAP1. Recognizes KEAP1 from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC
Description: A polyclonal antibody against KEAP1. Recognizes KEAP1 from Human, Mouse. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC; Recommended dilution: WB:1:200-1:1000, IHC:1:20-1:200
Description: A polyclonal antibody against Keap1. Recognizes Keap1 from Human, Mouse. This antibody is Unconjugated. Tested in the following application: ELISA, WB; Recommended dilution: WB:1:500-1:2000
Description: KEAP1 Antibody: KEAP1 (kelch-like ECH-associated protein 1) is a stress sensing adaptor for the Cullin3 (Cul3)-dependent E3 ubiquitin ligase complex that negatively regulates NRF2 (NF-E2-related factor 2) and plays a role in the oxidative stress response. It targets NFE2L2/NRF2 for ubiquitination and degradation by the proteasome. KEAP1 contains an amino terminal BTB/POZ domain and a carboxyl terminal KELCH domain which are required for interaction with NRF2, and in binding Cul3-E3 ubiquitin ligase. Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD). KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation. Mutation of the KEAP1 gene is found in lung cancer.
Description: KEAP1 Antibody: KEAP1 (kelch-like ECH-associated protein 1) is a stress sensing adaptor for the Cullin3 (Cul3)-dependent E3 ubiquitin ligase complex that negatively regulates NRF2 (NF-E2-related factor 2) and plays a role in the oxidative stress response. It targets NFE2L2/NRF2 for ubiquitination and degradation by the proteasome. KEAP1 contains an amino terminal BTB/POZ domain and a carboxyl terminal KELCH domain which are required for interaction with NRF2, and in binding Cul3-E3 ubiquitin ligase. Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD). KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation. Mutation of the KEAP1 gene is found in lung cancer.
Description: Can be used for various proteomics studies in both normal and pathological cases. It is an excellent control and suitable for educational purposes. This product is prepared from whole tissue homogenates and has undergone SDS-PAGE quality control analysis. The protein is stored in a buffer with protease inhibitor cocktail fo prevent degradation.
Description: Can be used for various proteomics studies in both normal and pathological cases. It is an excellent control and suitable for educational purposes. This product is prepared from whole tissue homogenates and has undergone SDS-PAGE quality control analysis. The protein is stored in a buffer with protease inhibitor cocktail fo prevent degradation.
Description: Description of target: Acts as a substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1 and targets NFE2L2/NRF2 for ubiquitination and degradation by the proteasome, thus resulting in the suppression of its transcriptional activity and the repression of antioxidant response element-mediated detoxifying enzyme gene expression. Retains NFE2L2/NRF2 and may also retain BPTF in the cytosol. Targets PGAM5 for ubiquitination and degradation by the proteasome.<p>This subsection of the <a href="//www.uniprot.org/help/function_section">‘Function’</a> section describes the metabolic pathway(s) associated with a protein.<p><a href='/help/pathway' target='_top'>More...</a></p>Pathwayi: protein ubiquitinationThis protein is involved in the pathway protein ubiquitination, which is part of Protein modification._x000D_View all proteins of this organism that are known to be involved in the pathway protein ubiquitination and in Protein modification. ;Species reactivity: Rat;Application: ;Assay info: Assay Methodology: Quantitative Sanadwich ELISA;Sensitivity: 11.75 pg/mL
Description: Description of target: Acts as a substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1 and targets NFE2L2/NRF2 for ubiquitination and degradation by the proteasome, thus resulting in the suppression of its transcriptional activity and the repression of antioxidant response element-mediated detoxifying enzyme gene expression. Retains NFE2L2/NRF2 and may also retain BPTF in the cytosol. Targets PGAM5 for ubiquitination and degradation by the proteasome.5 Publications
<p>Manually curated information for which there is published experimental evidence.</p>
<p><a href="/manual/evidences#ECO:0000269">More…</a></p> Manual assertion based on experiment iniRef.7"Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress."_x005F_x005F_x000D_Zhang D.D., Hannink M._x005F_x005F_x000D_Mol. Cell. Biol. 23:8137-8151(2003) [PubMed] [Europe PMC] [Abstract]Cited for: FUNCTION, MUTAGENESIS OF CYS-151; CYS-273 AND CYS-288, SUBCELLULAR LOCATION.Ref.8"Fetal Alz-50 clone 1 interacts with the human orthologue of the Kelch-like Ech-associated protein."_x005F_x005F_x000D_Strachan G.D., Morgan K.L., Otis L.L., Caltagarone J., Gittis A., Bowser R., Jordan-Sciutto K.L._x005F_x005F_x000D_Biochemistry 43:12113-12122(2004) [PubMed] [Europe PMC] [Abstract]Cited for: INTERACTION WITH NF2L2/NRF2 AND BPTF, FUNCTION, TISSUE SPECIFICITY, SUBCELLULAR LOCATION.Ref.9"Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex."_x005F_x005F_x000D_Zhang D.D., Lo S.-C., Cross J.V., Templeton D.J., Hannink M._x005F_x005F_x000D_Mol. Cell. Biol. 24:10941-10953(2004) [PubMed] [Europe PMC] [Abstract]Cited for: FUNCTION, INTERACTION WITH CUL3 AND RBX1, MUTAGENESIS OF 125-ILE--GLY-127 AND 162-TYR--ILE-164, UBIQUITINATION.Ref.10"Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway."_x005F_x005F_x000D_Zhang D.D., Lo S.C., Sun Z., Habib G.M., Lieberman M.W., Hannink M._x005F_x005F_x000D_J. Biol. Chem. 280:30091-30099(2005) [PubMed] [Europe PMC] [Abstract]Cited for: FUNCTION, IDENTIFICATION IN A COMPLEX WITH CUL3 AND RBX1, UBIQUITINATION, ENZYME REGULATION, MUTAGENESIS OF CYS-151.Ref.13"PGAM5, a Bcl-XL-interacting protein, is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex."_x005F_x005F_x000D_Lo S.-C., Hannink M._x005F_x005F_x000D_J. Biol. Chem. 281:37893-37903(2006) [PubMed] [Europe PMC] [Abstract]Cited for: INTERACTION WITH PGAM5, FUNCTION, ENZYME REGULATION, DOMAIN, MUTAGENESIS OF CYS-151; TYR-334; ARG-415; ARG-483 AND TYR-572. ;Species reactivity: Human;Application: ELISA;Assay info: Assay Methodology: Quantitative Sandwich Immunoassay;Sensitivity: < 0.056 ng/mL
Description: Description of target: Acts as a substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1 and targets NFE2L2/NRF2 for ubiquitination and degradation by the proteasome, thus resulting in the suppression of its transcriptional activity and the repression of antioxidant response element-mediated detoxifying enzyme gene expression. Retains NFE2L2/NRF2 and may also retain BPTF in the cytosol. Targets PGAM5 for ubiquitination and degradation by the proteasome (By similarity).By similarity2 Publications
<p>Manually curated information for which there is published experimental evidence.</p>
<p><a href="/manual/evidences#ECO:0000269">More…</a></p> Manual assertion based on experiment iniRef.1"Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain."_x005F_x005F_x000D_Itoh K., Wakabayashi N., Katoh Y., Ishii T., Igarashi K., Engel J.D., Yamamoto M._x005F_x005F_x000D_Genes Dev. 13:76-86(1999) [PubMed] [Europe PMC] [Abstract]Cited for: NUCLEOTIDE SEQUENCE [MRNA], FUNCTION.Ref.6"Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression."_x005F_x005F_x000D_McMahon M., Itoh K., Yamamoto M., Hayes J.D._x005F_x005F_x000D_J. Biol. Chem. 278:21592-21600(2003) [PubMed] [Europe PMC] [Abstract]Cited for: FUNCTION. <p>Describes the metabolic pathway(s) associated with a protein.</p><p><a href='../manual/pathway' target='_top'>More...</a></p>Pathwayi: protein ubiquitinationThis protein is involved in the pathway protein ubiquitination, which is part of Protein modification._x005F_x005F_x000D_View all proteins of this organism that are known to be involved in the pathway protein ubiquitination and in Protein modification. ;Species reactivity: Mouse;Application: ELISA;Assay info: Assay Methodology: Quantitative Sandwich Immunoassay;Sensitivity: < 0.055 ng/mL
Description: Description of target: This gene encodes a protein containing KELCH-1 like domains, as well as a BTB/POZ domain. Kelch-like ECH-associated protein 1 interacts with NF-E2-related factor 2 in a redox-sensitive manner and the dissociation of the proteins in the cytoplasm is followed by transportation of NF-E2-related factor 2 to the nucleus. This interaction results in the expression of the catalytic subunit of gamma-glutamylcysteine synthetase. Two alternatively spliced transcript variants encoding the same isoform have been found for this gene.;Species reactivity: Human;Application: ELISA;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 40.6pg/mL
Description: A polyclonal antibody for detection of Keap1 from Human, Mouse, Rat. This Keap1 antibody is for WB, IHC-P, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the Internal region of human Keap1
Description: A polyclonal antibody for detection of Keap1 from Human, Mouse, Rat. This Keap1 antibody is for WB, IHC-P, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the Internal region of human Keap1
Description: A polyclonal antibody for detection of Keap1 from Human, Mouse, Rat. This Keap1 antibody is for WB, IHC-P, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the Internal region of human Keap1
Description: A synthetic peptide for use as a blocking control in assays to test for specificity of KEAP1 antibody, catalog no. 20R-1096
×
Identification of novel fibronectin-binding proteins by 2D-far Westernblot in atypical enteropathogenic Escherichia coli serotype O55:H7
Atypical enteropathogenic Escherichia coli (aEPEC) is a subgroup of EPEC, which is among the main pathogens liable for deadly diarrhoea in youngsters. In contrast with typical EPEC (tEPEC), aEPEC lack an EAF (EPEC adherence issue) plasmid (pEAF), which encodes a sequence of virulence-associated genes.
The extracellular matrix (ECM) element of human cells has been reported to be an vital component within the interplay between host and bacterial pathogens. On this analysis, a 2D-Far Western blot technique was carried out to identifiy the bacterial proteins that might bind to fibronectin, one of the vital frequent constituents of ECM. A complete of 17 protein spots had been recognized, together with Four outer membrane proteins (OMPs), specifically, OmpC, OmpD, OmpX and LamB. In vitro research had been used to find out whether or not these OMPs had been concerned within the adherence course of.
By way of oblique immunofluorescence assays, 4 OMPs could possibly be noticed on the surfaces of host cells. After incubating the cells with the recombinant proteins, the adhesion fee of the O55:H7 isolate was decreased. Moreover, the deletion of OmpX and LamB also can lower the adhesion fee of WT. Taken collectively, a high-throughput screening technique for host ECM-binding proteins primarily based on 2D Far-Western blot was established, and 4 outer membrane proteins recognized by this technique had been discovered to be concerned within the adherence course of.
Validation of superior reference genes for qRT-PCR and Westernblot analyses in marine Emiliania huxleyi – virus mannequin system
Goals: To seek for a set of reference genes for dependable gene expression evaluation within the globally vital marine coccolithophore Emiliania huxleyi -virus mannequin system.
Strategies and outcomes: Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) had been evaluated for his or her stability as potential reference genes for qRT-PCR utilizing ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software program. CDKA, TUA and TUB genes had been examined as loading controls for Western blot in the identical pattern panel. Moreover, goal genes related to cell apoptosis, i.e., metacaspase genes, had been utilized to validate the collection of reference genes.
The evaluation outcomes demonstrated that putative housekeeping genes exhibited vital variations in each mRNA and protein content material throughout virus an infection. After a complete evaluation with all the algorithms, CDKA and GAPDH had been beneficial as probably the most secure reference genes for E huxleyi virus (EhV) an infection remedies. For Western blot, vital variation was seen for TUA and TUB, whereas CDKA was stably expressed, in keeping with the outcomes of qRT-PCR.
Conclusions: CDKA and GAPDH are the only option for gene and protein expression evaluation than the opposite candidate reference genes below EhV an infection situations.
Questioning protection values decided by 2D WesternBlots – a essential examine on the characterization of anti-HCP ELISA reagents
Host cell proteins (HCPs) represent a serious class of process-related impurities, whose substantial clearance should be demonstrated by appropriate analytical strategies to warrant product high quality and cut back potential security dangers for sufferers. On this regard, enzyme linked immunosorbent assays (ELISAs), which primarily depend on the standard of the HCP reference normal (immunogen) and HCP-specific polyclonal antibodies, are thought of the gold normal for HCP monitoring.
For the qualification of the employed antibodies, 2D Western Blots (2D-WBs) are the popular approach to find out the protection, although quite a lot of sensible constraints are properly acknowledged. By utilizing a number of orthogonal approaches, corresponding to affinity-based mass spectrometry and oblique ELISA, the current examine revealed potential detection gaps (i.e., non-covered HCPs) of typical 2D-WBs, which could be primarily attributed to two completely different root causes: i) low quantities of proteins or antibodies being unable to beat the detection restrict and ii) western blot artifacts as a result of lack of conformational epitopes via protein denaturation hindering HCP-antibody recognition.
In distinction, the shortage of particular antibodies towards sure (significantly, low molecular weight) HCPs, as proposed in earlier research, appears to play solely a minor position. Collectively, these findings suggest that CHO-HCP ELISA antibodies are higher than qualification research by 2D-WBs point out. This text is protected by copyright. All rights reserved.
