Journal: Scientific Reports

Article Title: GATS tag system is compatible with biotin labelling methods for protein analysis

doi: 10.1038/s41598-023-36858-y

Figure Lengend Snippet: Identification of anti-GAMA rabbit monoclonal antibodies. ( a ) A simplified cellular model of Plasmodium falciparum . ( b ) Domain information on the GAMA protein and the location of GAMA-F. ( c ) Image showing immunoblotting evaluation of the binding of antibodies to GAMA-F and Ripr-F (control) synthesized using a wheat cell-free protein synthesis system. Antibodies are unpurified supernatants produced in Expi293F cells. ( d ) In vitro interaction assay of rabbit monoclonal antibodies clone (Ra) and GAMA-F. Ripr-F is used as control. Antibodies are unpurified supernatants produced in Expi293F cells. All AlphaScreen signals are raw luminescent signals in the AlphaScreen-based biochemical assay. Error bars denote the standard deviation (independent experiments; n = 3). ( e ) Evaluation of antibody specificity with lysates of P. falciparum . Antibodies are produced in Expi293F cells and purified by protein G Sepharose 4 Fast Flow (GE Healthcare).

Article Snippet: Antibody binding was detected using an AlphaScreen IgG (Protein A) detection kit (Perkin Elmer).

Techniques: Bioprocessing, Western Blot, Binding Assay, Control, Synthesized, Produced, In Vitro, Amplified Luminescent Proximity Homogenous Assay, Standard Deviation, Purification

Journal: Scientific Reports

Article Title: GATS tag system is compatible with biotin labelling methods for protein analysis

doi: 10.1038/s41598-023-36858-y

Figure Lengend Snippet: Broad epitope determination of Ra3, Ra9, and Ra13. ( a ) Diagram representing the GAMA-F fragment for epitope determination. ( b ) Identification of epitope region using A, B, and C GAMA-F fragments. These fragments were fused to the N-terminus of GST-TEV-bls protein and synthesized using a wheat germ cell-free system. Binding between the fusion protein and Ra3, Ra9, and Ra13 antibodies was detected by AlphaScreen. ( c ) Identification of epitope region using smaller segmented fragments of A, B, and C. ( d ) Diagram representing the lysine residues of the GAMA-F fragment.

Article Snippet: Antibody binding was detected using an AlphaScreen IgG (Protein A) detection kit (Perkin Elmer).

Techniques: Synthesized, Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Scientific Reports

Article Title: GATS tag system is compatible with biotin labelling methods for protein analysis

doi: 10.1038/s41598-023-36858-y

Figure Lengend Snippet: The minimal amino acid epitope determination of Ra3. ( a ) Immunoblot analysis of deletion mutants to identify the minimum Ra48 epitope sequence. ( b ) Kinetics assay of the GATS tag and anti-GATS antibody. Anti-GATS antibody was captured on a protein G-immobilized Biacore sensorchip at 1500 RU. Purified FLAG-GST-GATS protein was then injected for 1600 s as analyte. Black lines represent a global fit of a 1:1 interaction model to each kinetic data set. ( c ) Pointing out antibody concentrations in AlphaScreen. The GATS tag was fused to the N-terminus of GST-TEV-bls protein and synthesized using a wheat germ cell-free system.

Article Snippet: Antibody binding was detected using an AlphaScreen IgG (Protein A) detection kit (Perkin Elmer).

Techniques: Western Blot, Sequencing, Purification, Injection, Amplified Luminescent Proximity Homogenous Assay, Synthesized

Journal: eLife

Article Title: Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

doi: 10.7554/eLife.83477

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , AlphaScreen SureFire pERK 1/2 (Thr202/Tyr204) Assay Kits , PerkinElmer , , .

Techniques: Purification, Expressing, Plasmid Preparation, Recombinant, Dominant Negative Mutation, Amplified Luminescent Proximity Homogenous Assay, Software

Journal: Molecular cancer therapeutics

Article Title: A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-?B

doi: 10.1158/1535-7163.MCT-11-0362

Figure Lengend Snippet: (A) The phosphorylation status of I-κBα was evaluated using AlphaScreen pIκB assay and by (B) western blot analysis in A549 cells following 6-hour treatment with 0, 1, 3, and 6 μM NSC 47147. Results show a decrease in luminescence indicative of pIκB inhibition. Western blot confirms decreasing levels of pIκB proteins and total I-κBα proteins in response to NSC 47147. (C) TNFα induced pIκB levels after 6-hour treatment with and without NSC 47147. A549 cells were stimulated with 10 ng/ml TNFα following pre-treatment with 6 μM NSC 47147. Cellular lysates were subjected to analysis by pIκB AlphaScreen and western blot.

Article Snippet: Phospho-IκB levels were monitored in whole cell lysates using AlphaScreen SureFire p-IκB (S32/36) assay kit, (PerkinElmer, Shelton, CT), a homogenous bead-based assay designed to measure the phosphorylation of endogenous p-IκB (S32/36) in cell lysates, according to the manufacturer’s protocol.

Techniques: Phospho-proteomics, Amplified Luminescent Proximity Homogenous Assay, Western Blot, Inhibition