Patched Protein Detection
Patched Protein Detection - Related Videos
09:00
Detection of Protein Ubiquitination
0 Views •
Cited by 96 •
2009
Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
09:43
Patch Clamp Electrophysiology
0 Views •
2023
Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply suction to form a high resistance seal. This process isolates a small “patch” of membrane that contains one or more ion channels.
03:41
Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions
0 Views •
2025
In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.
07:42
In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces
0 Views •
Cited by 7 •
2010
This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.
11:42
Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies
0 Views •
Cited by 42 •
2015
The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.
FAQs
The key interpretive link is the relationship between Patched, Hedgehog, and Smoothened. Hedgehog binding relieves Patched-mediated inhibition of Smoothened, so changes in Patched abundance or distribution can be examined as part of pathway regulation. Detection results therefore help researchers relate receptor patterns to altered Hedgehog signaling in developmental or experimental systems.
Patched localization adds spatial information that total protein measurements may not provide. Immunofluorescence and immunohistochemistry can show where Patched appears within cells or tissue sections, allowing researchers to compare receptor distribution across developing regions. Those spatial differences can support analyses of tissue patterning and cell differentiation, where location is biologically relevant.
Immunoblotting is suited to assessing Patched abundance, whereas immunofluorescence and immunohistochemistry additionally reveal subcellular or tissue localization. Using these readouts addresses complementary questions: whether the amount of detected protein changes and where that protein is situated. Together, they provide a broader view of Patched expression and distribution in an experimental sample.
A typical workflow begins with cells or tissue sections, followed by antibody recognition of Patched. A fluorescent or enzyme-linked label then makes the antibody-bound protein detectable. The resulting signal is examined according to the selected method, either for localization in cells or tissue sections or for protein abundance by immunoblotting.
This analysis is useful when researchers need to connect Hedgehog pathway regulation with developmental outcomes. Comparing Patched expression or distribution across an experimental system can help characterize processes involved in tissue patterning and cell differentiation. The measurements provide molecular evidence that complements observations of developmental organization or changes in cell fate.
Altered Patched expression or distribution can provide evidence of changed Hedgehog activity in disease models and other experimental systems. In developmental biology, this supports investigations of congenital abnormalities by linking receptor patterns with pathway regulation. The same measurements can also help compare normal and affected tissues or assess how experimental conditions influence developmental signaling.