Abstract:
:Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Stumptner C,Gogg-Kamerer M,Viertler C,Denk H,Zatloukal Kdoi
10.1016/bs.mie.2015.08.003subject
Has Abstractpub_date
2016-01-01 00:00:00pages
139-62eissn
0076-6879issn
1557-7988pii
S0076-6879(15)00455-3journal_volume
568pub_type
杂志文章abstract::White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several nonadipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, nonadipocyte cell p...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-411619-1.00003-3
更新日期:2014-01-01 00:00:00
abstract::The development of sensors that mimic the natural smell sensing mechanism and selectively recognizes the odorants remains highly challenging. Electrochemical based sensing approaches aiming at monitoring molecular recognition events between surface receptors and analytes in solution or in the gas phase, are one possib...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.04.071
更新日期:2020-01-01 00:00:00
abstract::Cholestatic syndromes are inborn or acquired disorders of bile formation. In recent years, several inherited cholestatic syndromes were characterized at the molecular level: progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC). Both PFIC and BRIC were divided phenot...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)00031-5
更新日期:2005-01-01 00:00:00
abstract::It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.080
更新日期:2017-01-01 00:00:00
abstract::As for other GPCRs, the oligomerization of glycoprotein hormone receptors (GPHRs) appears as critical event for receptor function. By means of modern techniques based on the BRET or FRET principle, GPHR oligomerization has been reported to explain several physiological and pathological conditions. In particular, the p...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391862-8.00020-X
更新日期:2013-01-01 00:00:00
abstract::Glycoproteins are a major class of glycoconjugates displaying a variety of mutual interactions between glycan and protein moieties that ultimately affect molecular organization. Modulation of the pendant glycan structures is important in tuning the functions of glycoproteins. Here we discuss structural aspects and som...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)78018-6
更新日期:2010-01-01 00:00:00
abstract::Specific recognition of mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Structural determination of the eIF4E-cap complexes, as well as complexes of eIF4E with other proteins regulating its activity, requires complementary experiments that allow for energetic an...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)30009-8
更新日期:2007-01-01 00:00:00
abstract::Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.08.024
更新日期:2016-01-01 00:00:00
abstract::A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The id...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.008
更新日期:2016-01-01 00:00:00
abstract::Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recom...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04049-8
更新日期:2005-01-01 00:00:00
abstract::The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.09.028
更新日期:2016-01-01 00:00:00
abstract::Retinoic acid (RA), the biologically active metabolite of vitamin A, regulates a vast spectrum of biological processes, such as cell differentiation, proliferation, apoptosis, and morphogenesis. microRNAs (miRNAs) play a crucial role in regulating gene expression by binding to messenger RNA (mRNA) which leads to mRNA ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.02.009
更新日期:2020-01-01 00:00:00
abstract::The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constant...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(94)39023-1
更新日期:1994-01-01 00:00:00
abstract::Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397945-2.00010-X
更新日期:2013-01-01 00:00:00
abstract::The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Severa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391860-4.00013-6
更新日期:2012-01-01 00:00:00
abstract::Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(87)39079-2
更新日期:1987-01-01 00:00:00
abstract::In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate fast...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-420037-1.00008-7
更新日期:2013-01-01 00:00:00
abstract::Three-dimensional (3D) cryoelectron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Databank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB), and the N...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)83004-6
更新日期:2010-01-01 00:00:00
abstract::Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11009-5
更新日期:2006-01-01 00:00:00
abstract::Isothermal titration calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of binding reactions. In addition, it enables label-free measurement of enzymatic reactions. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit valid...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.023
更新日期:2016-01-01 00:00:00
abstract::Hypoxia and inflammation are coincidental events in an array of diseased tissues, including chronically inflamed sites (e.g., inflammatory bowel disease, rheumatoid arthritis), growing tumors, myocardial infarcts, atherosclerotic plaques, healing wounds, and sites of bacterial infection (Murdoch et al., 2005). An unde...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)35025-8
更新日期:2007-01-01 00:00:00
abstract::The analysis of a reaction pathway by stopped-flow and chemical quench-flow methods provides the only means of directly measuring the rates of reaction at the active site of an enzyme. The interpretation of data from each technique is similar and involves solution of the differential equations describing the approach ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)34129-6
更新日期:1986-01-01 00:00:00
abstract::Formation of a Lys48-linked polyubiquitin chain is required for destruction of targeted proteins by the 26S proteasome, whereas formation of a Lys63-linked polyubiquitin chain is required for modulation of protein-protein interaction, enzyme activity, and intracellular localization. In addition, monoubiquitination pla...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)99006-X
更新日期:2005-01-01 00:00:00
abstract::Cytoskeletal motors utilize the energy stored in ATP to generate linear motion along rigid filaments. Because their enzymatic cycles are tightly coupled to the production of force and forward movement, the optical-trapping technique is uniquely suited for studying their mechanochemical cycle. Here, we discuss the prac...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.037
更新日期:2017-01-01 00:00:00
abstract::Acoustic sensors probe the response of a thin layer to the mechanical displacement associated with an acoustic wave. Acoustic measurements provide two simultaneous time-resolved signals; one signal is related to the velocity or frequency of the acoustic wave and is mainly a function of adsorbed mass, while the second ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)65002-3
更新日期:2009-01-01 00:00:00
abstract::Extracellular signals dictate the biological processes of neural stem cells (NSCs) both in vivo and in vitro. The intracellular response elicited by these signals is dependent on the context in which the signal is received, which in turn is decided by previous and concurrent signals impinging on the cell. A synthesis ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)19002-3
更新日期:2006-01-01 00:00:00
abstract::The hypoxia-inducible factor (HIF) is a heterodimeric basic helix-loop-helix (bHLH) transcription factor that controls the mammalian cellular transcriptional response to low oxygen tension by up-regulating genes including glycolytic enzymes and angiogenic factors, such as the vascular endothelial growth factor (VEGF)....
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)35005-2
更新日期:2007-01-01 00:00:00
abstract::Genome sequencing projects have provided the substrate for an unimaginable number of biological experiments. Further, genomic technologies such as microarrays and quantitative and exquisitely sensitive techniques such as real-time quantitative polymerase chain reaction have made it possible to reliably generate millio...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05012-5
更新日期:2009-01-01 00:00:00
abstract::The three major considerations in reducing metal-gel binding are (1) the type of gel to be used, (2) the ionic strength of the eluent, and (3) alkaline reduction of the gel. Each of these parameters should be considered in advance of an experiment. A method is offered for determining the optimal gel type, gel treatmen...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(88)58044-8
更新日期:1988-01-01 00:00:00
abstract::The number of genetically modified mouse lines has been increasing exponentially in the past few decades. In order to safeguard them from accidental loss and genetic drifting, to reduce animal housing cost, and to efficiently distribute them around the world, it is important to cryopreserve these valuable genetic reso...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)76004-3
更新日期:2010-01-01 00:00:00