Solute service (SLC) transporters symbolize the second-largest fraction of the membrane proteome after G-protein-coupled receptors, however have been underutilized as drug targets and the operate of many members of this household continues to be unknown.
They’re technically difficult to work with as they’re tough to precise and extremely dynamic, making them unstable in detergent resolution. Many SLCs lack recognized inhibitors that could possibly be utilized for stabilization. Moreover, as they bind their physiological substrates with excessive micromolar to low millimolar affinities, binding and transport assays have confirmed to be notably difficult to implement.
nycodenz medium
Beforehand, we reported a GFP-based technique for the overexpression and purification of membrane proteins in Saccharomyces cerevisiae. Right here, we prolong this expression platform with the GFP thermal shift (GFP-TS) assay, which is a simplified model of fluorescence-detection size-exclusion chromatography that mixes the pattern versatility of fluorescence-detection size-exclusion chromatography with the high-throughput functionality of dye-based thermal shift assays.
We reveal how GFP-TS can be utilized for detecting particular ligand interactions of SLC transporter fusions and measuring their affinities in crude detergent-solubilized membranes. We additional present how GFP-TS might be employed on purified SLC transporter fusions to display screen for particular lipid-protein interactions, which is a vital complement to native mass spectrometry approaches that can’t cope simply with crude lipid-mixture preparations.
This protocol is easy to carry out and might be adopted by researchers with a fundamental background in protein chemistry. Beginning with an SLC transporter assemble that may be expressed and purified from S. cerevisiae in a well-folded state, this protocol extension might be accomplished in ~4-5 d.
Thermostability-based binding assays reveal advanced interaction of cation, substrate and lipid binding within the bacterial DASS transporter, VcINDY
The divalent anionsodium symporter (DASS) household of transporters (SLC13 household in people) are key regulators of metabolic homeostasis, disruption of which ends up in safety from diabetes and weight problems, and inhibition of liver most cancers cell proliferation.
Thus, DASS transporter inhibitors are enticing targets within the therapy of persistent, age-related metabolic illnesses. The characterisation of a number of DASS transporters has revealed variation within the substrate selectivity and suppleness within the coupling ion used to energy transport.
Right here, utilizing the mannequin DASS co-transporter, VcINDY from Vibrio cholerae, we’ve got examined the interaction of the three main interactions that happen throughout transport: the coupling ion, the substrate, and the lipid atmosphere.
Utilizing a collection of high-throughput thermostability-based interplay assays, we’ve got proven that substrate binding is Na+-dependent; a requirement that’s orchestrated by way of a mixture of electrostatic attraction and Na+-induced priming of the binding website structure.
Now we have recognized novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carboxylate teams within the ligand which are exactly distanced to fulfill carboxylate interplay areas of the substrate binding website. Now we have additionally recognized a posh relationship between substrate and lipid interactions, which suggests a dynamic, regulatory position for lipids in VcINDY’s transport cycle.
| Rat Cholesterol ELISA ELISA | |||
| BlueGene | |||
| Goat Cholesterol ELISA ELISA | |||
| BlueGene | |||
| Mouse Cholesterol ELISA ELISA | |||
| BlueGene | |||
Liposome Flotation Assay for Learning Interactions Between Rubella Virus Particles and Lipid Membranes
Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that’s pathogenic to people. RuV binds to the goal cell through the viral envelope protein E1, however the particular receptor molecules on the goal cell are but to be totally elucidated.
Right here, we describe a protocol for liposome flotation assay to check direct interactions between RuV particles and lipid membranes in a qualitative method. Interactions are examined by a Nycodenz density gradient fractionation utilizing UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids.
Fractionated RuV particles are detected utilizing normal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) adopted by Western blot evaluation for viral proteins. On the Axis Shield Nycodenz gradient, RuV particles sure to liposomes shift to decrease density fractions than unbound RuV particles.
Utilizing this protocol, we offer compelling proof that, at impartial pH in a calcium-dependent method, RuV particles bind to lipid membranes containing each sphingomyelin (SM) and ldl cholesterol in sure cell sorts.
| Rat Cholesterol ELISA ELISA | |
| E01A11128 | BlueGene |
| Goat Cholesterol ELISA ELISA | |
| E01A46041 | BlueGene |
| Mouse Cholesterol ELISA ELISA | |
| E01A19869 | BlueGene |
| Human Cholesterol ELISA ELISA | |
| E01A2368 | BlueGene |
| Sheep Cholesterol ELISA ELISA | |
| E01A98335 | BlueGene |
Detection of Salmonid IgM Particular to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Utilizing Lipid-Modified Antigens in a Bead-Based mostly Antibody Detection Assay.
Bead-based multiplex immunoassays are promising instruments for dedication of the particular humoral immune response. On this examine, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies towards Piscine orthoreovirus (PRV).
Three completely different genotypes of PRV (PRV-1, PRV-2, and PRV-3) trigger illness in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a bunch receptor binding protein and a goal for neutralizing and protecting antibodies.
Whereas recombinant σ1 carried out poorly as an antigen to detect particular antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled delicate detection of particular IgM within the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma.
Binding of non-specific IgM to beads coated with management antigens additionally elevated after PRV an infection, indicating a launch of polyreactive antibodies. This non-specific binding was lowered by warmth therapy of plasma. The identical immunoassay additionally detected anti-PRV-Three σ1 antibodies from contaminated rainbow trout.
In abstract, a refined bead primarily based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed delicate detection of anti-PRV-1 and anti-PRV-Three antibodies from salmonids.
| Rat Cholesterol ELISA ELISA | |||
| E01A11128 | |||
| Goat Cholesterol ELISA ELISA | |||
| E01A46041 | |||
| Mouse Cholesterol ELISA ELISA | |||
| E01A19869 | |||
Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays.
Membrane fusion is an important course of within the eukaryotic cell. Specialised proteins are essential to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We element right here a way for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography.
Learning fusion reactions in vitro requires purified fusion proteins to be inserted right into a lipid bilayer. Liposomes are splendid mannequin membranes, as lipid composition and dimension could also be adjusted. To this finish, we describe a reconstitution technique by detergent elimination for Drosophila atlastin into preformed liposomes.
Whereas a number of reconstitution strategies can be found, reconstitution by detergent elimination has a number of benefits that make it appropriate for atlastins and different comparable proteins. The benefit of this technique features a excessive reconstitution yield and proper orientation of the reconstituted protein.
Lipid Peroxide (LPO) Assay Kit |
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| abx294113-50g | Abbexa | 50 µg | Ask for price |
Lipid Peroxidation (LPO)Assay Kit |
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| BC015-50T | ELK Biotech | 50T | EUR 200 |
Lipid Peroxidation (MDA) Assay Kit |
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| abx096010-100Assays | Abbexa | 100 Assays | EUR 453.6 |
Lipid Peroxidation (LPO) Assay Kit |
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| abx096010-100l | Abbexa | 100 µl | EUR 425 |
Lipid Peroxidation (LPO) Assay Kit |
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| abx096010-1ml | Abbexa | 1 ml | Ask for price |
Lipid Peroxidation (LPO) Assay Kit |
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| abx096010-200l | Abbexa | 200 µl | Ask for price |
Lipid Peroxidation (MDA) Assay Kit |
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| GWB-AXR340 | GenWay Biotech | 100 assays | Ask for price |
Lipid Peroxidation MDA Assay Kit |
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| EGQA0070 | EnoGene | 100T | EUR 85 |
Lipid Peroxidation (MDA) Assay Kit |
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| K2167-100 | ApexBio | 100 assays | EUR 51.2 |
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Description: Cell Biology|RNS detection |
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Lipid Peroxidation (MDA) Assay Kit |
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| K2167-500 | ApexBio | 500T | EUR 198.4 |
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Description: Cell Biology|RNS detection |
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Lipid Peroxidation (MDA) Assay kit |
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| MBS9718963-48Tests | MyBiosource | 48Tests | EUR 160 |
Lipid Peroxidation (MDA) Assay kit |
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| MBS9718963-5x96Tests | MyBiosource | 5x96Tests | EUR 745 |
Lipid Peroxidation (MDA) Assay kit |
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| MBS9718963-96Tests | MyBiosource | 96Tests | EUR 170 |
Lipid Peroxidation Colorimetric Assay Kit |
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| K454-100 | Biovision | each | EUR 652.8 |
Lipid Peroxide -LPO- Fluorometric Assay Kit |
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| E-BC-F003-96T | Elabscience Biotech | 96T | EUR 426 |
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Description: Cell-based (quantitative) |
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Lipid Peroxide -LPO- Fluorometric Assay Kit |
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| E-BC-F003-each | Elabscience Biotech | each | Ask for price |
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Description: Cell-based (quantitative) |
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Lipid Peroxide -LPO- Colorimetric Assay Kit |
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| E-BC-K176-M-48T | Elabscience Biotech | 48T | EUR 26 |
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Description: Quantitative |
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Lipid Peroxide -LPO- Colorimetric Assay Kit |
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| E-BC-K176-M-96T | Elabscience Biotech | 96T | EUR 380 |
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Description: Quantitative |
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Lipid Peroxide -LPO- Colorimetric Assay Kit |
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| E-BC-K176-M-each | Elabscience Biotech | each | Ask for price |
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Description: Quantitative |
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Lipid Peroxidation Assay, Catalog: MA-0102 |
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| MA-0102 | Akrivis Bio | 100 wells | EUR 385 |
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Description: A plate-based colorimetric assay to measure malondialdehyde (MDA) levels in samples |
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Lipid Bilayer Permeability Disruption Assay Kit |
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| BPD100K | ProFoldin | 100 assays | EUR 354.89 |
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Description: This product includes 1.25 ml of liposomal dye, 1.25 ml of color development solution, 7.5 ml of buffer and 1 ml of DMSO. It is for assays of 100 samples using a 96-well plate. |
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Lipid peroxidation assay kit (Colorimetric method) |
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| MBS2540553-48Test | MyBiosource | 48Test | EUR 325 |
Lipid peroxidation assay kit (Colorimetric method) |
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| MBS2540553-48Tests | MyBiosource | 48Tests | EUR 325 |
Lipid peroxidation assay kit (Colorimetric method) |
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| MBS2540553-5x96Tests | MyBiosource | 5x96Tests | EUR 1900 |
Lipid peroxidation assay kit (Colorimetric method) |
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| MBS2540553-96Test | MyBiosource | 96Test | EUR 415 |
Lipid peroxidation assay kit (Colorimetric method) |
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| MBS2540553-96Tests | MyBiosource | 96Tests | EUR 415 |
Peroxide Quantitative Assay Kit (Lipid-Compatible) |
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| BSP067 | Bio Basic | 250Assays, 250preps | EUR 110.11 |
CheKine™ Micro Lipid Peroxidation (MDA) Assay kit |
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| KTB1050-48T | Abbkine | 48 T | EUR 39 |
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Description: Abbkine CheKine™ Micro Lipid Peroxidation (MDA) Assay kit is designed for the quantitative determination of lipid peroxidation. |
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CheKine™ Micro Lipid Peroxidation (MDA) Assay kit |
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| KTB1050-96T | Abbkine | 96 T | EUR 49 |
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Description: Abbkine CheKine™ Micro Lipid Peroxidation (MDA) Assay kit is designed for the quantitative determination of lipid peroxidation. |
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CheKine™ Micro Lipid Peroxidation (MDA) Assay kit |
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| KTB1050-each | Abbkine | each | Ask for price |
Lipid Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit |
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| K739-100 | Biovision | each | EUR 698.4 |
Cell Meterâ„¢ Fluorimetric Cellular Lipid Peroxidation Assay Kit |
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| 22906-200Tests | AAT Bioquest | 200 Tests | EUR 334 |
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Description: Lipid peroxidation is the oxidative degradation of cellular lipid by reactive oxygen species (ROS). |
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This technique might be prolonged to different membrane proteins and for different purposes that require proteoliposomes. Moreover, we describe a FRET primarily based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.
| MULTIPLEX KIT PCR MASTITIS PCR kit | |
| PCR-MPX218-48D | Bioingentech |
| MULTIPLEX KIT PCR MASTITIS PCR kit | |
| PCR-MPX218-96D | Bioingentech |
| MULTIPLEX KIT PCR Babesia & Theileria PCR kit | |
| PCR-MPX401-48D | Bioingentech |
| MULTIPLEX KIT PCR Babesia & Theileria PCR kit | |
| PCR-MPX401-96D | Bioingentech |
| Leaf PCR Kit | |
| 11140007-1 | Glycomatrix |
Nycodenz MSDS
