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Identification of Proteins Using Four Methods of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
摘要： An upper-division undergraduate- or graduate-level laboratory experiment, challenging students to identify bovine serum albumin, equine myoglobin, bovine calmodulin, and bovine cytochrome c, uses four methods of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): linear positive, in-source decay, peptide mass fingerprint, and postsource decay. Individual hands-on experience with each method is provided in a three week experiment. The importance of sequencing proteins/peptides when identifying proteomic targets using MALDI-TOF MS is emphasized.
关键词： Mass Spectrometry,Hands-On Learning/Manipulatives,Upper-Division Undergraduate,Laboratory Instruction,Graduate Education/Research,Bioanalytical Chemistry,Biotechnology,Analytical Chemistry,Biochemistry
更新于2025-09-19 17:13:59
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The effect of low level laser irradiation on oxidative stress, muscle damage and function following neuromuscular electrical stimulation. A double blind, randomised, crossover trial
摘要： Background: Low level laser therapy (LLLT) is among novel methods for preventing and treating muscle damage and soreness induced by volitional exercise, but little is known about using LLLT before neuromuscular electrical stimulation. The aim of this first randomised, double blind, crossover trial addressing this issue was to evaluate effects of LLLT on muscle damage and oxidative stress, as well as recovery of muscle function after a single session of isometric neuromuscular electrical stimulation(NMES). Methods: Twenty four moderately active, healthy men aged 21–22 years received 45 electrically evoked tetanic, isometric contractions of the quadriceps femoris, preceded by LLLT or sham-LLLT. Maximal isometric voluntary muscle torques, perceived soreness, and blood samples were analysed from baseline to 96 h post intervention. We measured plasma markers of muscle damage (the activity of creatine kinase), and inflammation (C-reactive protein), and evaluated redox state parameters. Results: NMES-evoked contractions induced oxidative stress, demonstrated by an increase in lipid peroxidation and impairments in enzymatic antioxidant system. LLLT irradiations had a protective effect on NMES-induced decrease in enzymatic antioxidant defence and shortened the duration of inflammation. This effect of irradiations on redox state and inflammation did not affect lipid peroxidation, muscle damage, and muscle torque. Conclusions: LLLT may protect from impairments in enzymatic antioxidant system and may shorten inflammation induced by a single NMES session in moderately active, healthy men. However, the effects of LLLT on redox state and inflammatory processes do not seem to affect muscle damage and recovery of muscle function after NMES.
关键词： Photobiomodulation,Evoked contractions,Knee extensors,DOMS,Biochemistry,Exercise-induced muscle damage
更新于2025-09-16 10:30:52
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Chemical Nanoplasmonics: Emerging Interdisciplinary Research Field at Crossroads between Nanoscale Chemistry and Plasmonics
摘要： Plasmonics research deals with understanding and manipulating the interaction between light and matter at a scale that is significantly smaller than the wavelength of light (e.g., metal nanoparticles). Such an interaction can be correlated with various forms of energy and signals such as thermal energy and optical spectra. Research efforts in plasmonics range from rationally designing and precisely synthesizing nanostructures that allow for unraveling and reliably tuning novel and useful plasmonic properties (e.g., surface-enhanced spectroscopies and photothermal effects) to ultimately obtaining and utilizing plasmonic functionalities for applications, for example, in the biomedical field. With enormous potential and versatility in terms of plasmonic materials and devices, the principles of plasmonics are expected to provide new or improved solutions to many important challenges in various subfields of chemistry, including nanoparticle chemistry, catalytic reactions, surface-enhanced Raman scattering, photovoltaics, sensing, biochemistry, and therapeutics. Additionally, many hurdles and issues related to the advances and applications of plasmonics can be addressed by material- or property-based chemistry at the nanoscale (i.e., nanochemistry), while chemical principles and methods can offer new research directions in plasmonics. Nanochemistry allows scientists to develop exquisitely accurate methods for the synthesis of nanostructures with high precision and provides tools for functionalizing and analyzing complex plasmonic nanostructures (e.g., heterostructured-nanoparticles). Therefore, recent advancements in nanochemistry with plasmonic materials have made a great impact on the proper use and real applications of plasmonics, and plasmonics offers in turn new pathways and tools for chemical processes. The field of chemical nanoplasmonics includes the study of nanoscale chemistry for the advancement of plasmonics and the use of plasmonics to address key issues and challenges in chemistry. Newly emerging principles, methods, and materials in plasmonics can be useful in various fields of chemistry, including optical and chemical hot spots, typically based on strong electromagnetic fields formed within plasmonic nanostructures, as well as single-molecule and 3D SERS with plasmonic hot-spot platforms. Plasmonic multimers (e.g., gold nanoparticle dimers), plasmonic supercrystals, plasmonic nanoparticle lattices, gold nanobipyramids, virus-sized gold nanorods, spherical nucleic acids, plasmonic metamaterials, and chiral plasmonic structures are some of the key nanostructures for materials chemistry-based plasmonics. Hot charge carriers and plasmon-driven catalysis have been identified as important directions for many subfields of chemistry including physical chemistry, materials chemistry, and catalysis. Further, newly emerging platforms such as plasmonic nanoparticle-interfaced cell membranes, DNA origami-based plasmonics, and graphene-based nonlinear plasmonics have emerged as next-generation platforms that can provide new ways of forming functional materials and devices, including optical and computing devices.
关键词： photovoltaics,plasmonics,nanochemistry,biochemistry,nanoplasmonics,sensing,surface-enhanced Raman scattering,therapeutics
更新于2025-09-12 10:27:22
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Chemical cross-linking of a variety of green fluorescent proteins as F?rster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience
摘要： F?rster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and dif?culty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green-emitting ?uorescent proteins (FPs) as donors for a cross-linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses.
关键词： Upper-division undergraduate,orange ?uorescent protein,?uorescence lifetimes,proteins,green ?uorescent protein,biochemistry,TCSPC,?uorescence spectroscopy
更新于2025-09-10 09:29:36
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VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy
摘要： Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.
关键词： biochemistry,fluorescence microscopy,coiled coil,chemical biology,electron microscopy
更新于2025-09-09 09:28:46
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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
摘要： A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
关键词： Issue 142,cell-cell adhesion,fluorescence correlation spectroscopy,cell-cell interactions,Protein-protein interactions,number and brightness,fluorescence fluctuation spectroscopy,Biochemistry,N&B
更新于2025-09-09 09:28:46