Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor & alpha; and Specificity Protein 1 with Composite DNA Elements
This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor & ndash;DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Gold Nanoparticle-Based F & ouml;rster Resonance Energy Transfer (FRET) Analysis of Estrogen Receptor: DNA Interaction
Estrogen receptors play critical roles in regulating genes responsible for development and maintenance of reproductive tissues and other physiological function. The interaction of ERs with DNA sequences, known as estrogen response elements (EREs) (a palindromic repeat separated by three-base spacer, 5 & prime;GGTCAnnnTGACC-3 & prime;), is required for estrogen regulation of target gene expression. Here, we describe a simple & ldquo;mix-and-measure & rdquo;-based method for detecting ER:ERE interactions using ERE-immobilized metal nanoparticles and water-soluble conjugated polyelectrolytes (CPEs) as cooperative sensing elem...
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
In Situ Hybridization of Estrogen Receptors & alpha; and & beta; and GPER in the Human Testis
In situ hybridization (ISH) is an excellent method for detecting RNA in histological sections, both to detect gene expression and to assign gene expression to a distinct cell population. Therefore, ISH may be used in basic cell biology to detect the expression of certain genes within a tissue containing various cell populations. Here, we describe the detection and cellular localization of three estrogen receptors, both isoforms of the genomic estrogen receptor (ER & alpha; and ER & beta;) as well as the membrane-bound G-protein-coupled estrogen receptor 1 (GPER) in the human testis. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Erratum to: Methods for Analysis of AP-3/Rabin4 & rsquo; in Regulation of Lysosome Distribution
(Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - June 16, 2015 Category: Biochemistry Source Type: news
Identification of the Rab5 Binding Site in p11 & beta;: Assays for PI3K & beta; Binding to Rab5
Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for ...
Source: Springer protocols feed by Biochemistry - June 16, 2015 Category: Biochemistry Source Type: news
Methods for Analysis of AP-3/Rabin4 & rsquo; in Regulation of Lysosome Distribution
The position of lysosomes in the cytoplasm is important for their ability to fuse with the plasma membrane and release of proteases that are involved in tissue remodeling. Motor-directed bidirectional transport along microtubules is a critical process determining the distribution of lysosomes. How lysosomes are tethered to microtubules is incompletely understood, but a role for small GTPases of rab and arl families has been documented. We recently found that the rab5 and rab4 effector rabip4′ interacts with the adaptor complex AP-3 in a rab4-dependent manner on tubular endosomes. We here describe the assays that led ...
Source: Springer protocols feed by Biochemistry - June 16, 2015 Category: Biochemistry Source Type: news
Analysis of Connecdenn 1 & ndash;3 (DENN1A-C) GEF Activity for Rab35
Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that direct...
Source: Springer protocols feed by Biochemistry - June 16, 2015 Category: Biochemistry Source Type: news
Erratum to: Methods for Analysis of AP-3/Rabin4’ in Regulation of Lysosome Distribution
(Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - April 25, 2015 Category: Biochemistry Source Type: news
Analysis of the Interactions Between Rab GTPases and Class V Myosins
We present here a detailed description of this yeast two-hybrid “living chip” assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Measuring Rab GTPase-Activating Protein (GAP) Activity in Live Cells and Extracts
Mammalian cells encode a diverse set of Rab GTPases and their corresponding regulators. In vitro biochemical screening has proven invaluable in assigning particular Rabs as substrates for their cognate GTPase-activating proteins. However, in vitro activity does not always reflect substrate specificity in cells. This method describes a functional test of GAP activity in cells or extracts that takes into account the presence of other factors or conditions that might change observed in vitro specificity. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins
Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format ba...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Rab-NANOPS: FRET Biosensors for Rab Membrane Nanoclustering and Prenylation Detection in Mammalian Cells
Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane ancho...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Bioinformatic Approaches to Identifying and Classifying Rab Proteins
The bioinformatic annotation of Rab GTPases is important, for example, to understand the evolution of the endomembrane system. However, Rabs are particularly challenging for standard annotation pipelines because they are similar to other small GTPases and form a large family with many paralogous subfamilies. Here, we describe a bioinformatic annotation pipeline specifically tailored to Rab GTPases. It proceeds in two steps: first, Rabs are distinguished from other proteins based on GTPase-specific motifs, overall sequence similarity to other Rabs, and the occurrence of Rab-specific motifs. Second, Rabs are classified takin...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein
Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein–dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quan...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy
Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot–Marie–Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF sign...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
