Springer protocols feed by Molecular Medicine 
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.
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.Protein Quantitation of the Developing Cochlea Using Mass Spectrometry
Mass spectrometry-based proteomics allows for the measurement of hundreds to thousands of proteins in a biological system. Additionally, mass spectrometry can also be used to quantify proteins and peptides. However, observing quantitative differences between biological systems using mass spectrometry-based proteomics can be challenging because it is critical to have a method that is fast, reproducible, and accurate. Therefore, to study differential protein expression in biological samples labeling or label-free quantitative methods can be used. Labeling methods have been widely used in quantitative proteomics, however labe...
Source: Springer protocols feed by Molecular Medicine - January 1, 2016 Category: Molecular Biology Source Type: news
Multiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory Research
Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a compl...
Source: Springer protocols feed by Molecular Medicine - January 1, 2016 Category: Molecular Biology Source Type: news
Yeast Two-Hybrid Screening to Test for Protein & ndash;Protein Interactions in the Auditory System
We describe a protocol to screen for protein–protein interactions using the Gal-4-based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method gives the most number of true interactions. Where a premade library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick a...
Source: Springer protocols feed by Molecular Medicine - January 1, 2016 Category: Molecular Biology Source Type: news
The Single-Molecule Approach to Membrane Protein Stoichiometry
The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background...
Source: Springer protocols feed by Molecular Medicine - January 1, 2016 Category: Molecular Biology Source Type: news
Helios & reg; Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates
The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher & rsquo;s skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios & reg; Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coa...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Inner-Ear Proteins
Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered, and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molec...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays
The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microar...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
Generation of Noninvasive, Quantifiable, Orthotopic Animal Models for NF2-Associated Schwannoma and Meningioma
Schwannomas and meningiomas are nervous system tumors that can occur sporadically or in patients with neurofibromatosis type 2 (NF2). Mutations of the Neurofibromatosis 2 (NF2) gene are frequently observed in these tumors. Schwannomas and meningiomas cause significant morbidities, and an FDA-approved medical therapy is currently not available. The development of preclinical animal models that accurately capture the clinical characteristics of these tumors will facilitate the evaluation of novel therapeutic agents for the treatment of these tumors, ultimately leading to more productive clinical trials. Here, we describe the...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear
There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to ...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation
The avian embryo has a well-documented history as a model system for the study of neurogenesis, morphogenesis, and cell fate specification. This includes studies of the chicken inner ear that employ in ovo electroporation, in conjunction with the Tol2 system, to yield robust long-term transgene expression. Capitalizing on the success of this delivery method, we describe a modified version of the Tol2 expression vector that readily accepts the insertion of a microRNA-encoding artificial intron. This offers a strategy to investigate the possible roles of different candidate microRNAs in ear development by overexpression. Her...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
Helios® Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates
The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher’s skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios® Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated ...
Source: Springer protocols feed by Molecular Medicine - December 31, 2015 Category: Molecular Biology Source Type: news
Qualitative and Quantitative Analysis of Histone Deacetylases in Kidney Tissue Sections
Fluorescent microscope imaging technologies are increasing in their applications and are being used on a wide scale. However methods used to quantify the level of fluorescence intensity are often not utilized—perhaps given the result may be immediately seen, quantification of the data may not seem necessary. However there are a number of reasons given to quantify fluorescent images including the importance of removing potential bias in the data upon observation as well as quantification of large numbers of images gives statistical power to detect subtle changes in experiments. In addition discreet localization of a p...
Source: Springer protocols feed by Molecular Medicine - December 22, 2015 Category: Molecular Biology Source Type: news
Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation
Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuc...
Source: Springer protocols feed by Molecular Medicine - December 22, 2015 Category: Molecular Biology Source Type: news
Laser Capture Microdissection of Archival Kidney Tissue for qRT-PCR
Whole-organ molecular analysis of the kidney potentially misses important factors involved in the pathogenesis of disease in glomeruli and tubules. Organ wide analysis can however be augmented by using laser capture microdissection (LCM) to isolate morphologically similar cells and nephron structures from a heterogeneous tissue section via direct visualization of the cells. The protocol here provides a practical approach utilizing LCM in combination with RNA isolation techniques for downstream analysis. This technique is readily applicable to study mRNA expression in isolated glomeruli and tubules in both experimental anim...
Source: Springer protocols feed by Molecular Medicine - December 22, 2015 Category: Molecular Biology Source Type: news
Combining Near Infrared Fluorescent Imaging for Calcification and Inflammation in Vascular Tissue Samples Ex Vivo
Quantification of vascular lesions in models of vascular calcification is crucial for testing novel treatments, but remains a challenging endeavor. Conventional methods include Oil-Red-O staining of whole tissue samples, calcium quantification in incinerated samples, or stereoisometric histologic processing. While most techniques offer fairly high levels of reliability, all of them share the fact that samples are not available for other assays following the analysis, as tissue is altered or destroyed in the course of the procedure. Furthermore, none is capable of measuring both calcification and inflammation at the same ti...
Source: Springer protocols feed by Molecular Medicine - December 22, 2015 Category: Molecular Biology Source Type: news
