In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of α-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.
24 Related JoVE Articles!
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)
Institutions: Louisiana State University Health Sciences Center.
Genetic screens conducted using Drosophila melanogaster
(fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila
GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo
, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1
to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.
Biochemistry, Issue 82, Drosophila, GAL4/UAS system, transgenic, Tandem Affinity Purification, protein-protein interaction, proteomics
Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Institutions: Ohio University, Russ College of Engineering and Technology, Ohio University, Brigham and Women's Hospital, Harvard Medical School.
Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc
.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.
Bioengineering, Issue 83, affinity chromatography, membrane adsorber, bioseparations, protein A, galectin-1, Gal-1hFc
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining
Institutions: CNRS UMR 5534, Université de Lyon 1, LabEX DEVweCAN, CNRS UPR 3296, CNRS UMR 5286.
Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ
in animal models. We describe a DNA-fluorescent in situ
hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.
Neuroscience, Issue 83, Life Sciences (General), Virology, Herpes Simplex Virus (HSV), Latency, In situ hybridization, Nuclear organization, Gene expression, Microscopy
Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI
Institutions: King Abdulaziz University, The University of Sheffield.
The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ
assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1
. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3
. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1
of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5
. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3
Immunology, Issue 93, Allergy, Immunology, IgE, Fcε, RI, horse (Equus caballus), Immunoassay
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Institutions: University of Akron.
Recombinant protein engineering has utilized Escherichia coli (E. coli)
expression systems for nearly 4 decades, and today E. coli
is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing a T7 lac
inducible vector and E. coli
expression hosts, starting from transformation to scale-up and purification.
Bioengineering, Issue 83, protein engineering, recombinant protein production, AviTag, BirA, biotinylation, pET vector system, E. coli, inclusion bodies, Ni-NTA, size exclusion chromatography
Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants
Institutions: RWTH Aachen University, Fraunhofer Institute for Molecular Biology and Applied Ecology.
Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei
Cel5A, in Nicotiana tabacum
are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei
Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.
Environmental Sciences, Issue 88, heterologous expression, endoplasmic reticulum, endoglucanase, cellulose, glycosyl-hydrolase, fluorescence, cellulase, Trichoderma reesei, tobacco plants
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Institutions: RWTH Aachen University.
-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for S
of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGA
T-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for m
ransfer of A
roups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.
Biochemistry, Issue 93, S-adenosyl-l-methionine, AdoMet, SAM, aziridine cofactor, double activated cofactor, methyltransferase, DNA methylation, protein methylation, biotin labeling, fluorescence labeling, SMILing, mTAG
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors
Institutions: Technion - Israel Institute of Technology, Technion - Israel Institute of Technology, Technion - Israel Institute of Technology.
A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli
K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.
by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a 'real time' observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104
cells/ml) in less than an hour.
Bioengineering, Issue 81, analytical chemistry, silicon materials, microbiology, optical materials, Porous Si, optical biosensor, bacteria detection, label-free biosensor, nanostructure, E. coli bacteria
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Institutions: GE Healthcare Bio-Sciences AB.
In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280
. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka
) and affinity (KD
), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.
Cellular Biology, Issue 37, Protein interaction, Surface Plasmon Resonance, Biacore X100, CFCA, Cystatin B, Papain
Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
Institutions: Georgia Institute of Technology .
The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1
. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1
to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fcγ receptors with IgG Fc1-6
, selectins with glycoconjugate ligands6-9
, integrins with ligands10-13
, homotypical cadherin binding14
, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5
, membrane anchor2
, molecular orientation and length6
, carrier stiffness9
, and impingement force20
, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4
, and trimolecular interactions19
using a modified model21
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17
. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin αL
on neutrophils with dimeric E-selectin in the solution to activate αL
Bioengineering, Issue 57, Two-dimensional binding, affinity and kinetics, micropipette manipulation, receptor-ligand interaction
Determining the Phagocytic Activity of Clinical Antibody Samples
Institutions: Ragon Institute of MGH, MIT, and Harvard, Dartmouth College.
Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6
. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7
. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 μm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of FcγRs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9
Immunology, Issue 57, Phagocytosis, Antibody, ADCC, Effector Function, Fc receptor, antibody-dependent phagocytosis, monocytes
A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
Institutions: University of Alberta, Universidad Nacional Autónoma de México, Universidad Nacional Autónoma de México.
Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.
Immunology, Issue 74, Biochemistry, Infection, Cellular Biology, Molecular Biology, Medicine, Neutrophils, Neutrophil, Monocyte, PMN, NF- κB, ERK, integrin, Signal Transduction, inflammation, flow cytometry, immunolabeling, nuclear factors, cytokines, cells, assay
Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
Institutions: State University of New York Upstate Medical University.
gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli
. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.
Biochemistry, Issue 76, Genetics, Molecular Biology, Cellular Biology, Microbiology, Bacteria, Proteins, Mgm101, Rad52, mitochondria, recombination, mtDNA, maltose-binding protein, MBP, E. coli., yeast, Saccharomyces cerevisiae, chromatography, electron microscopy, cell culture
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
Institutions: The Rockefeller University.
To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p
-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.
Genetics, Issue 79, Receptors, G-Protein-Coupled, Protein Engineering, Signal Transduction, Biochemistry, Unnatural amino acid, site-directed mutagenesis, G protein-coupled receptor, targeted photocrosslinking, bioorthogonal labeling, targeted epitope tagging
Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Institutions: Royal Institute of Technology.
Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1
. To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2
. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3
. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4
, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5
, a small, bispecific molecule with affinity for both HSA and the novel target was identified6
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli
with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7
Molecular Biology, Issue 59, Affinity chromatography, albumin-binding domain, human serum albumin, Z-domain
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Institutions: Mayo Clinic.
Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.
Cellular Biology, Issue 46, immunoprecipitation, flow cytometry, protein-protein interaction, multiprotein complex
Depletion of Specific Cell Populations by Complement Depletion
Institutions: Blood Research Institute.
The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1
. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37°C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.
JoVE Immunology, Issue 36, rabbit, complement, cell isolation, cell depletion