Springer protocols feed by Protein Science 
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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.Studying N-Linked Glycosylation of Receptor Tyrosine Kinases
Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membran...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Quantification of the Effects of Mutations on Receptor Tyrosine Kinase (RTK) Activation in Mammalian Cells
Single amino acid mutations in receptor tyrosine kinases (RTKs) are known to cause receptor over-activation and disease. Here we present a detailed protocol for the quantification of the effect of mutations on RTK activation in mammalian cells. The activation measurements are based on Western blotting, and involve direct comparison of receptor phosphorylation under conditions that ensure identical expression of wild-type and mutant receptors. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Analysis of Receptor Tyrosine Kinase Gene Amplification on the Example of FGFR1
FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Applying the Proximity Ligation Assay (PLA) to Mouse Preimplantation Embryos for Identifying Protein-Protein Interactions In Situ
Analysis of protein-protein interactions in mouse preimplantation embryos is hindered by the low cell number of the embryo. Here we describe the use of the proximity ligation assay to overcome these limitations and outline how protein-protein interactions can be visualized in situ. The method is based on a normal immunofluorescence labeling protocol of preimplantation embryos. Events of binding of the two primary antibodies directed against two individual proteins close to each other are visualized. If the two primary antibodies and the corresponding oligo-linked secondary antibodies bind in close proximity a cascade of ev...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Evaluation of the Dimerization Profiles of HER Tyrosine Kinases by Time-Resolved Förster Resonance Energy Transfer (TR-FRET)
Activation of receptor tyrosine kinases (RTK), such as those belonging to the human epidermal growth factor receptor (HER) family, occurs only after receptor dimerization, which is a crucial step for cellular signal transduction and diversification. The HER family includes four members (EGFR/HER1, HER2, HER3, and HER4) that can homodimerize or heterodimerize. Here, we describe immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET) to profile EGFR-EGFR, HER2-HER2, and EGFR-HER2 dimers directly in tumor samples. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Single-Molecule Optical Methods Analyzing Receptor Tyrosine Kinase Activation in Living Cells
We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Analysis of Epidermal Growth Factor Receptor Dimerization by BS3 Cross-Linking
Dimerization of receptor tyrosine kinases is a well-characterized process. It is imperative for the activation of many receptors, including the epidermal growth factor receptor (EGFR). EGFR has been shown to be regulated by a number of factors, including lipid raft localization. For example, alteration of the lipid raft localization of EGFR has been demonstrated to modify receptor dimerization. This protocol describes an assay to quantify EGFR dimers using BS3 cross-linking. BS3 cross-linking is well suited for this purpose because of its length, water solubility, and membrane impermeability. Although this protocol is writ...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Analysis of Changes in Phosphorylation of Receptor Tyrosine Kinases: Antibody Arrays
Tyrosine kinases are mainly classified into two groups, as receptor tyrosine kinase (RTK) and non-receptor tyrosine kinase (NRTK). The RTK family of transmembrane ligand-binding proteins are important mediators of the signaling cascade and includes EGFR, PDGFR (platelet-derived growth factor receptors), FGFR (fibroblast growth factor receptor) and the IR (insulin receptor). RTKs comprise 59 members and their structure includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain possessing the tyrosine kinase activity. This chapter focuses on antibody arrays that are basically used to...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Analysis of Receptor Tyrosine Kinase (RTK) Phosphorylation by Immunoblotting
Immunoblotting for phosphorylated forms of receptor tyrosine kinases (RTKs) has been the mainstay of investigations on RTK signaling for the past two decades. Despite the development of quantitative mass spectrometry, reverse-phase protein array, and multiplex technologies, immunoblotting with phospho-specific antibodies is still used in parallel with these technologies and remains a powerful, and reproducible, method for interrogating signaling networks involving RTKs. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2
Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. (Source: Springer prot...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Expression, Purification, and Immobilization of Recombinant Tamavidin 2 Fusion Proteins
Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Rescuing Aggregation-Prone Proteins in Escherichia coli with a Dual His6-MBP Tag
Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway® recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be dige...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
SUMO as a Solubility Tag and In Vivo Cleavage of SUMO Fusion Proteins with Ulp1
Expression of proteins in E. coli is often plagued by insolubility of the protein of interest. A solution to this problem is the expression of proteins as fusions to solubility tags such as the SUMO protein. SUMO fusion proteins can be cleaved to remove the SUMO moiety using SUMO-specific proteases such as Ulp1. Here, we describe the use of vectors for the expression of recombinant proteins in E. coli as fusions to the Drosophila SUMO protein. This includes a vector that encodes not only the SUMO tagged protein of interest but also SUMO-tagged Ulp1. Coexpression of these two proteins results in the in vivo cleavage of the ...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Simplified Protein Purification Using an Autoprocessing, Inducible Enzyme Tag
The development of affinity tags has greatly simplified protein purification procedures. A variety of affinity tags are now available to improve expression, solubility, and/or tag removal. In this chapter, we describe a method for purifying recombinant proteins expressed in Escherichia coli that uses a highly specific, inducible, C-terminal autoprocessing protease tag. This method streamlines affinity purification, cleavage, and tag separation into a one-step purification procedure, avoiding the need to remove fusion tags from target proteins with exogenous proteases. In addition to accelerating protein purification, we sh...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Purification of E. coli Proteins Using a Self-Cleaving Chitin-Binding Affinity Tag
The use of affinity tags to purify recombinant proteins is ubiquitous in molecular biology. However, tag removal after purification still remains a challenge. The most commonly used method, proteolytic digestion, has several drawbacks that make the process complex and costly. One alternative to the use of proteolytic digestion is the use of self-cleaving purification tags. Here, we describe a system that combines a chitin-binding domain (CBD) tag with the ∆I-CM intein to yield a self-cleaving purification tag. A protein gene of interest is genetically fused downstream of the tag, generating a fusion protein that can ...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
