The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters

The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters
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

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.
Custom Antibody titration by ELISA up to 2 rabbits and 1 bleed
ELISA-1
Beta2-Microglobulin ELISA kit ELISA Kit
LF-EK60047
Chicken thrombomodulin,TM ELISA KIT ELISA
QY-E80092

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.
Custom Antibody titration by ELISA up to 2 rabbits and 1 bleed
Alpha Diagnostics
Beta2-Microglobulin ELISA kit ELISA Kit
Abfrontier
Chicken thrombomodulin,TM ELISA KIT ELISA
Qayee Biotechnology

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.
Nycodenz, 500 grams
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.
Custom Antibody titration by ELISA up to 2 rabbits and 1 bleed
ELISA-1 Alpha Diagnostics
Beta2-Microglobulin ELISA kit ELISA Kit
LF-EK60047 Abfrontier
Chicken thrombomodulin,TM ELISA KIT ELISA
QY-E80092 Qayee Biotechnology
Oxycodone ELISA
EK7130 BosterBio
Amphiphysin ELISA
LF-EK0189 Abfrontier

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).
The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters
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.
Custom Antibody titration by ELISA up to 2 rabbits and 1 bleed
ELISA-1
Beta2-Microglobulin ELISA kit ELISA Kit
LF-EK60047
Chicken thrombomodulin,TM ELISA KIT ELISA
QY-E80092

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.

Cell Meter™ Intracellular Colorimetric Lipid Peroxidation (MDA) Assay Kit

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Cell Navigator™ Fluorimetric Lipid Droplet Assay Kit *Green Fluorescence*

22730 200 Tests
EUR 262.8

Cell Navigator™ Fluorimetric Lipid Droplet Assay Kit *Red Fluorescence*

22735 500 Tests
EUR 262.8

Lipid A Lipopolysaccharide (LPS Lipid A / Endotoxin) Antibody

abx415762-1ml 1 ml
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Lipid A Antibody

abx023083-1ml 1 ml
EUR 886.8

Lipid Extraction & Polar/Neutral Lipid Separation Combo Kit (Chloroform Free)

MET-5009-C 50 preps
EUR 790.8
Description: This kit combines a chloroform free organic lipid extraction with further separation of polar and neutral lipids. The lipid extraction first extracts lipids from a crude lipid source to a chloroform free upper organic phase using a proprietary alcohol. After addition of a proprietary organic compound, the mixture is centrifuged to separate the phases. The recovered organic phase containing lipids is dried and then polar and neutral lipids are separated by adding a proprietary secondary alcohol and organic compound. The layers are separated, dried, and resuspended for further analysis.

Ceraplex Lipid Concentrate(1000X)

C3316-010 100ml
EUR 144

Ceraplex Lipid Concentrate(1000X)

C3316-050 500ml
EUR 295.2

Lipid Droplet Isolation Kit

MET-5011 50 preps
EUR 498
Description: Lipid droplets are lipid rich organelles found in the adipose tissue of eukaryotes. They function to regulate the hydrolysis and storage of neutral lipids and serve as storage for cholesterol and acyl-glycerols. Lipid droplets have also been associated with inflammatory responses, obesity, atherosclerosis, and cancer.

Lipid peroxidation inhibitor 1

HY-U00319 1mg
EUR 1448.4

Lipid Quantitation Kit (Colorimetric)

STA-613 100 assays
EUR 637.2
Description: Cell Biolabs? Lipid Quantification Kit measures the lipid content (unsaturated fatty acids only) of samples using the sulfo-phospho-vanillin method, resulting in a simple colorimetric readout amenable to multi-well plate detection.

Lipid Quantification Kit (Fluorometric)

STA-617 100 assays
EUR 637.2
Description: The Lipid Quantification Kit (Fluorometric) uses a fluorometric detection system to quantify neutral lipids in a convenient 96-well microplate reader. The kit includes sufficient reagents for 100 assays.

Anti-Lipid A antibody

STJ16100142 1 mL
EUR 346.8

Monophosphoryl Lipid A (Synthetic)

VAdv-Ly0012-1mg 1 mg
EUR 1033.2
Description: Synthetic Monophosphoryl Lipid A, pure compound, a TLR4-based vaccine adjuvant.

Monophosphoryl Lipid A (Synthetic)

VAdv-Ly0012-5mg 5 mg
EUR 3013.2
Description: Synthetic Monophosphoryl Lipid A, pure compound, a TLR4-based vaccine adjuvant.

Synthetic Monophosphoryl Lipid A

VAdv-Ly0031 1 mg
EUR 2557.2
Description: Synthetic monophosphoryl lipid A, a TLR4 agonist vaccine adjuvant.

LipidSpot 488 Lipid Droplet Stain

70065 125uL
EUR 481.2
Description: Minimum order quantity: 1 unit of 125uL

LipidSpot 488 Lipid Droplet Stain

70065-T 20uL
EUR 139.2
Description: Minimum order quantity: 1 unit of 20uL

LipidSpot 610 Lipid Droplet Stain

70069 125uL
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Description: Minimum order quantity: 1 unit of 125uL

LipidSpot 610 Lipid Droplet Stain

70069-T 20uL
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Description: Minimum order quantity: 1 unit of 20uL

Serum Albumin, Lipid Free Protein

20-abx260049
  • EUR 927.60
  • EUR 343.20
  • EUR 627.60
  • 100 mg
  • 10 mg
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Goat Lipid peroxide ELISA kit

E06L0263-192T 192 tests
EUR 1524
Description: A competitive ELISA for quantitative measurement of Goat Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Goat Lipid peroxide ELISA kit

E06L0263-48 1 plate of 48 wells
EUR 624
Description: A competitive ELISA for quantitative measurement of Goat Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Goat Lipid peroxide ELISA kit

E06L0263-96 1 plate of 96 wells
EUR 822
Description: A competitive ELISA for quantitative measurement of Goat Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Lipid peroxide ELISA kit

E04L0263-192T 192 tests
EUR 1524
Description: A competitive ELISA for quantitative measurement of Rabbit Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Lipid peroxide ELISA kit

E04L0263-48 1 plate of 48 wells
EUR 624
Description: A competitive ELISA for quantitative measurement of Rabbit Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Lipid peroxide ELISA kit

E04L0263-96 1 plate of 96 wells
EUR 822
Description: A competitive ELISA for quantitative measurement of Rabbit Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Human Lipid peroxide ELISA kit

E01L0263-192T 192 tests
EUR 1524
Description: A competitive ELISA for quantitative measurement of Human Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Human Lipid peroxide ELISA kit

E01L0263-48 1 plate of 48 wells
EUR 624
Description: A competitive ELISA for quantitative measurement of Human Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Human Lipid peroxide ELISA kit

E01L0263-96 1 plate of 96 wells
EUR 822
Description: A competitive ELISA for quantitative measurement of Human Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Mouse Lipid peroxide ELISA kit

E03L0263-192T 192 tests
EUR 1524
Description: A competitive ELISA for quantitative measurement of Mouse Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Mouse Lipid peroxide ELISA kit

E03L0263-48 1 plate of 48 wells
EUR 624
Description: A competitive ELISA for quantitative measurement of Mouse Lipid peroxide in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.
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.
PCR Mycoplasma Detection Kit
M034-Kit TOKU-E
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
Nycodenz MSDS

Nycodenz MSDS

Sources :
1. NCBI

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