Assay Kits Bile Acid Assay Fto Antibody Gels Glycodeoxycholic Acid Gsk3 Alpha Soft Agar Tgf Alpha Antibody
GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers
Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3.
Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.
Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology
Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms.
Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts
Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells.
The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.
Quantitative and Time-Resolved Monitoring of Organelle and Protein Delivery to the Lysosome with A Tandem Fluorescent Halo-GFP reporter
Lysosomal degradative compartments hydrolyze macromolecules to generate basic building blocks that fuel metabolic pathways in our cells. They also remove misfolded proteins and control size, function and number of cytoplasmic organelles via constitutive and regulated autophagy. These catabolic processes attract interest because their defective functioning is linked to human disease and their molecular components are promising pharmacologic targets. The capacity to quantitatively assess them is highly sought for. Here, we present a tandem-fluorescent reporter consisting of a HaloTag-GFP chimera appended at the C- or at the N-terminus of select polypeptides to monitor protein and organelle delivery to the lysosomal compartment.
The Halo-GFP changes color upon fluorescent pulse with cell-permeable HaloTag ligands and, again, upon delivery to acidic, degradative lysosomal compartments, where the fluorescent ligand-associated HaloTag is relatively stable, whereas the GFP portion is not, as testified by loss of the green fluorescence and generation of a protease-resistant, fluorescent HaloTag fragment. The Halo-GFP tandem fluorescent reporter presented in our study allows quantitative and, crucially, time-resolved analyses of protein and organelle transport to the lysosomal compartment by high resolution confocal laser scanning microscopy, antibody-free electrophoretic techniques and flow cytometry.
A direct-drive GFP reporter for studies of tracheal development in Drosophila
The Drosophila tracheal system consists of a widespread tubular network that provides respiratory functions for the animal. Its development, from ten pairs of placodes in the embryo to the final stereotypical branched structure in the adult, has been extensively studied by many labs as a model system for understanding tubular epithelial morphogenesis. Throughout these studies, a breathless (btl)-GAL4 driver has provided an invaluable tool to either mark tracheal cells during development or to manipulate gene expression in this tissue.
A distinct shortcoming of this approach, however, is that btl-GAL4 cannot be used to specifically visualize tracheal cells in the presence of other GAL4 drivers or other UAS constructs, restricting its utility. Here we describe a direct-drive btl-nGFP reporter that can be used as a specific marker of tracheal cells throughout development in combination with any GAL4 driver and/or UAS construct. This reporter line should facilitate the use of Drosophila as a model system for studies of tracheal development and tubular morphogenesis.
Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery
The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency.
GFP Expressing Human Glioblastoma Cells |
|||
| TR01-GFP | Neuromics | 500,000 Cells | 1624.8 EUR |
GFP Expressing Human Gastric Carcinoma N87 Cells |
|||
| TR02-GFP | Neuromics | 500,000 Cells | 1624.8 EUR |
GFP Expressing Human Renal Adenocarcinoma Cells (ACHN) |
|||
| TR04-GFP | Neuromics | 500,000 Cells | 1624.8 EUR |
GFP Expressing Human Prostate Carcinoma Cells (DU 145) |
|||
| TR03-GFP | Neuromics | 500,000 Cells | 1624.8 EUR |
Green Fluorescent Protein (GFP-fusion protein) ELISA Kit, 96 tests, Quantitative |
|||
| 800-420-GFP | Alpha Diagnostics | 1 kit | 854.4 EUR |
GFP |
|||
| GT22101 | Neuromics | 100 ul | 522 EUR |
GFP antibody |
|||
| 70R-10652 | Fitzgerald | 400 ul | 798 EUR |
GFP antibody |
|||
| 70R-12242 | Fitzgerald | 100 ug | 523.2 EUR |
GFP antibody |
|||
| 10R-8423 | Fitzgerald | 100 ul | 471.6 EUR |
GFP protein |
|||
| 30R-1302 | Fitzgerald | 100 ug | 333.6 EUR |
GFP Antibody |
|||
| 48671-100ul | SAB | 100ul | 399.6 EUR |
GFP Antibody |
|||
| 48671-50ul | SAB | 50ul | 286.8 EUR |
GFP Antibody |
|||
| 48704-100ul | SAB | 100ul | 399.6 EUR |
GFP Antibody |
|||
| 48704-50ul | SAB | 50ul | 286.8 EUR |
GFP Antibody |
|||
| 48705-100ul | SAB | 100ul | 399.6 EUR |
GFP Antibody |
|||
| 48705-50ul | SAB | 50ul | 286.8 EUR |
GFP Antibody |
|||
| 48776-100ul | SAB | 100ul | 399.6 EUR |
GFP Antibody |
|||
| 48776-50ul | SAB | 50ul | 286.8 EUR |
GFP Antibody |
|||
| 35538-100ul | SAB | 100ul | 302.4 EUR |
GFP antibody |
|||
| 20R-2897 | Fitzgerald | 100 ul | 471.6 EUR |
We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.