Functional analysis of CaSR mutants

Studies were performed in HEK293 cells maintained in DMEM-Glutamax media (Thermo Fisher) with 10% fetal bovine serum (Gibco) at 37ºC, 5% CO₂. Mutations were introduced into a construct expressing WT CaSR tagged with enhanced green fluorescent protein (pEGFP-N1-CaSR) by site-directed mutagenesis using the Quikchange Lightning Kit (Agilent Technologies) and gene-specific primers (Sigma Aldrich), as described (14 (link)). WT and mutant pEGFP-N1-CaSR constructs, and luciferase reporter constructs (pGL4.30–nuclear factor of activated T cells (NFAT) and pGL4.33–serum-response element [SRE], Promega) were transiently transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies) 48 hours before the experiments, as described (14 (link), 15 (link)). Successful transfection was confirmed by visualizing green fluorescent protein (GFP) fluorescence using an Eclipse E400 fluorescence microscope with an epifluorescence filter, and images were captured using a DXM1200C digital camera and NIS Elements software (Nikon) (11 (link), 14 (link), 15 (link)).

Free full text: Click here

Dharmaraj P., Gorvin C.M., Soni A., Nelhans N.D., Olesen M.K., Boon H., Cranston T., Thakker R.V, & Hannan F.M. (2020). Neonatal Hypocalcemic Seizures in Offspring of a Mother With Familial Hypocalciuric Hypercalcemia Type 1 (FHH1). The Journal of Clinical Endocrinology and Metabolism, 105(5), 1393-1400.

Publication 2020

DMEM-Glutamax media fetal bovine serum Quikchange Lightning Kit gene-specific primers Lipofectamine 2000 Eclipse E400 DXM1200C digital camera NIS Elements software

Camera Enhanced green fluorescent protein Fetal bovine serum Fluorescence Fluorescence microscope Gene Green fluorescent protein Hek293 cells Lipofectamine 2000 Luciferase Mutations Pgl4 Primers Promega Serum response element Site directed mutagenesis T cells factor Transfection

Corresponding Organization : University of Oxford

Other organizations : Alder Hey Children's NHS Foundation Trust, Wrexham Maelor Hospital, Churchill Hospital

Top 5 similar protocols

Variable analysis

independent variables

Mutations introduced into a construct expressing WT CaSR tagged with enhanced green fluorescent protein (pEGFP-N1-CaSR)

dependent variables

Activation of nuclear factor of activated T cells (NFAT) and serum-response element (SRE) signaling pathways

control variables

HEK293 cells maintained in DMEM-Glutamax media with 10% fetal bovine serum at 37ºC, 5% CO2
Transfection of WT and mutant pEGFP-N1-CaSR constructs, and luciferase reporter constructs (pGL4.30–NFAT and pGL4.33–SRE) into HEK293 cells

controls

WT pEGFP-N1-CaSR construct as a positive control

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!