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.
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.

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 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.

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.

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 Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit

K2167-100 100 assays
EUR 586

Cell Meter™ Fluorimetric Cellular Lipid Peroxidation Assay Kit

22906 200 Tests
EUR 306
  • R-phrase: R20, R21, R22
  • H-Phrase: H303, H313, H333
  • Symbol for dangerous compounds: Xn
  • UNSPEC Code: 12352200

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

22730 200 Tests
EUR 219
  • R-phrase: R20, R21, R22
  • H-Phrase: H303, H313, H333
  • Symbol for dangerous compounds: Xn
  • UNSPEC Code: 12352200

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

22735 500 Tests
EUR 219
  • R-phrase: R20, R21, R22
  • H-Phrase: H303, H313, H333
  • Symbol for dangerous compounds: Xn
  • UNSPEC Code: 12352200

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

15991 200 tests
EUR 393
  • R-phrase: R20, R21, R22
  • H-Phrase: H303, H313, H333
  • Symbol for dangerous compounds: Xn
  • UNSPEC Code: 12352200

Lipid A Lipopolysaccharide (LPS Lipid A / Endotoxin) Antibody

abx415762-1ml 1 ml
EUR 606
  • Shipped within 1 week.

Lipid A Antibody

abx023083-1ml 1 ml
EUR 739
  • Shipped within 5-10 working days.

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

MET-5009-C 50 preps
EUR 659
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 120

Ceraplex Lipid Concentrate(1000X)

C3316-050 500ml
EUR 246

Lipid peroxidation inhibitor 1

HY-U00319 1mg
EUR 1207

Lipid Droplet Isolation Kit

MET-5011 50 preps
EUR 415
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.

Monophosphoryl Lipid A (Synthetic)

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

Monophosphoryl Lipid A (Synthetic)

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

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

Lipid Quantitation Kit (Colorimetric)

STA-613 100 assays
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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 531
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 289

LipidSpot 488 Lipid Droplet Stain

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EUR 401
Description: Minimum order quantity: 1 unit of 125uL

LipidSpot 488 Lipid Droplet Stain

70065-T 20uL
EUR 116
Description: Minimum order quantity: 1 unit of 20uL

LipidSpot 610 Lipid Droplet Stain

70069 125uL
EUR 401
Description: Minimum order quantity: 1 unit of 125uL

LipidSpot 610 Lipid Droplet Stain

70069-T 20uL
EUR 114
Description: Minimum order quantity: 1 unit of 20uL

Rat Lipid peroxide ELISA kit

E02L0263-192T 192 tests
EUR 1270
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
Description: A competitive ELISA for quantitative measurement of Rat 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.

Rat Lipid peroxide ELISA kit

E02L0263-48 1 plate of 48 wells
EUR 520
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
Description: A competitive ELISA for quantitative measurement of Rat 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.

Rat Lipid peroxide ELISA kit

E02L0263-96 1 plate of 96 wells
EUR 685
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
Description: A competitive ELISA for quantitative measurement of Rat 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 1270
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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 520
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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 685
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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.

Mouse Lipid peroxide ELISA kit

E03L0263-192T 192 tests
EUR 1270
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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 520
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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

E03L0263-96 1 plate of 96 wells
EUR 685
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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.

Human Lipid peroxide ELISA kit

E01L0263-192T 192 tests
EUR 1270
  • Kit contents: 1. MICROTITER PLATE * 1 2. ENZYME CONJUGATE*1 vial 3. STANDARD A*1 vial 4. STANDARD B*1 vial 5. STANDARD C*1 vial 6. STANDARD D*1 vial 7. STANDARD E*1 vial 8. STANDARD F*1 vial 9. SUBSTRATE A*1 vial 10. SUBSTRATE B*1 vial 11. STOP SOLUT
  • Show more
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.
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.

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