TY - JOUR. T1 - Reversible DNA-Protein Cross-Linking at Epigenetic DNA Marks. AU - Ji, Shaofei. AU - Shao, Hongzhao. AU - Han, Qiyuan. AU - Seiler, Christopher L.. AU - Tretyakova, Natalia Y.. N1 - Funding Information: We thank Xun Ming and Peter Villalta (University of Minnesota) for their help with MS analysis and Robert Carlson (University of Minnesota) for preparing graphics for this manuscript. This research was supported by a grant from the NIEHS (ES023350). S.J. was partially supported by a Wayland E. Noland fellowship from the University of Minnesota. Publisher Copyright: © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. PY - 2017/11/6. Y1 - 2017/11/6. N2 - 5-Formylcytosine (5fC) is an endogenous DNA modification frequently found within regulatory elements of mammalian genes. Although 5fC is an oxidation product of 5-methylcytosine (5mC), the two epigenetic marks show distinct genome-wide distributions and protein affinities, suggesting that they perform different functions in ...
Homobifunctional crosslinkers possess identical reactive groups at opposite ends of the protein crosslinker s spacer arm, and can be reacted in a one-step chemical crosslinking reaction.
This invention is related to a thermoplastic crosslinked product obtainable by the crosslinking reaction of a composition comprising (A) a polymer having a silicon-containing group and (B) a tetravalent tin compound, said silicon-containing group having a hydrolyzable group bound to a silicon atom and capable of crosslinking through formation of a siloxane bond.
The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was ...
In this work, intramolecular and intermolecular associations under the effect of shear flow of dilute aqueous alkaline solutions of dextan, hydroxyethylcellulose (HEC), and the hydrophobically modified analogue (HM-2-HEC) in the presence of a chemical cross-linking agent were characterized with the aid of viscometry and rheo-small angle light scattering (rheo-SALS) methods. The picture that emerges at short times during the cross-linking reaction at a constant shear rate is that HEC coils contract because of intramolecular cross-linking; whereas the HM-2-HEC species show an incipient association and the dextran molecules are unaffected due to their compact structures. At longer times, interchain cross-linking of the polymer promotes the growth of large flocs, which are disrupted by shear forces when they are sufficiently large. The delicate interplay between intramolecular and intermolecular association is governed by factors such as the magnitude of the shear rate, the cross-linking agent ...
DNA interstrand cross-links (ICLs) covalently link the Watson and Crick strands of DNA and are extremely cytotoxic. Widely used chemotherapeutics (e.g. nitrogen...
BACKGROUND: The biological effects of CD24 (FL-80) cross-linking on breast cancer cells have not yet been established. We examined the impact of CD24 cross-linking on human breast cancer cell line MCF-7. METHODS: MCF-7 and MDA-MB-231 cells were treated with anti-rabbit polyclonal IgG or anti-human CD24 rabbit polyclonal antibodies to induce cross-linking, and then growth was studied. Changes in cell characteristics such as cell cycle modulation, cell death, survival in three-dimensional cultures, adhesion, and migration ability were assayed after CD24 cross-linking in MCF-7. RESULTS: Expression of CD24 was analyzed by flow cytometry in MDA-MB-231 and MCF-7 cells where 2% and 66% expression frequencies were observed, respectively. CD24 cross-linking resulted in time-dependent proliferation reduction in MCF-7 cells, but no reduction in MDA-MB-231 cells. MCF-7 cell survival was reduced by 15% in three-dimensional culture after CD24 cross-linking. Increased MCF-7 cell apoptosis was observed after ...
Dried hemoactive materials comprise both a cross-linked biologically compatible polymer and a non-cross-linked biologically compatible polymer. The cross-linked polymer is selected to form a hydrogel when exposed to blood. The non-cross-linked polymer is chosen to solubilize relatively rapidly when exposed to blood. The non-cross-linked polymer serves as a binder for holding the materials in desired geometries, such as sheets, pellets, plugs, or the like. Usually, the cross-linked polymer will be present in a particulate or fragmented form. The materials are particularly suitable for hemostasis and drug delivery.
ChemInform Abstract: Incorporating Disulfide Cross-Links at the Terminus of Oligonucleotides via Solid-Phase Nucleic Acid Synthesis.|span||/span| | S. E. OSBORNE; A. D. ELLINGTON | download | BookSC. Download books for free. Find books
This chapter reviews modern trends in the development of cross-linking approaches and their implication for studies of rRNA folding in the ribosome and the organization of ribosomal functional centers. It illustrates the results emanating from the ribosomal cross-linking studies with data taken from the authors' work on the investigation of the environment of mRNA and 5S rRNA in the ribosome. The application of a combination of short-range and long-range crosslinking reagents has proved to be a very fruitful strategy for investigation of the topography of RNA and proteins in the ribosome. A section briefly describes the photo-reactive compounds used in most cross-linking experiments with ribosomes. Trifluoromethylaryldiazirines (TFMAD)-containing photoreagents can be used for scanning very fast conformational changes within the ribosome structure. The data reviewed in the chapter show that site-specific photo-cross-linking is a powerful tool for the identification of interacting elements of the
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies. ...
The activation of G protein-coupled receptors (GPCR) mediates a variety of important biological signals. GPR110 belongs to the adhesion GPCR class with distinctively long N-terminus. Although GPR110 is an orphan receptor with unknown ligand, it has been identified as an oncogene implicated in lung and prostate cancers. In an effort to understand the molecular basis of GPR110 activation, we probed GPR110 conformation in living cells using chemical crosslinking and mass spectrometry. HEK cells overexpressing HA-GPR110 were incubated with disuccinimidyl suberate (DSS, a lysine-specific crosslinker). The DSS-modified HA-GPR110 was pulled-down and subjected to SDS-PAGE, tryptic digestion, and nanoLC-ESI-MS/MS. The MS-based approach identified 17 crosslinked lysine pairs in the N-terminal domain of GPR110. Among them, K29-K38, K187-K240, K240-K254, K398-442, K398-K438, K398-K427, K427-K442, K427-K438, and K151-K442, resulted from through-space crosslinking between two peptide segments, while K31-K32, ...
B. DNA-Protein Cross-Linking. To cross-link protein to the genomic DNA, 270 µl of formalin was added to 10 ml of DMEM containing acini to obtain a final solution of 1% formaldehyde. Cells were agitated for 10 minutes (Note 1) on a shaker table. Following the addition of 514 µl of 2.5 M glycine (125 mM final) for 5 minutes to quench the formaldehyde and terminate the crosslinking reaction, cells were centrifuged at 2000 rpm for 3 minutes at 40C. The pellet of acinar cells was washed twice with ice cold PBS.. C. Isolation of Nuclei from Isolated Acinar Cells. To isolate nuclei, acinar cells were incubated in Cytoplasmic Extract buffer (CE Buffer; 10 mM HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 0.1% Triton-X100, 0.5 mM DTT and protease inhibitors [5 µg/mL Aprotinin, 5 µg/mL Leupeptin, 5 µg/mL Pepstatin, 75 µg/mL PMSF]) followed by 5 strokes of Potter-Elvehjem homogenizer. The cells were set on ice for 15 minutes. To pellet the nuclei, the samples were centrifuged at 4oC and 5000 g for 10 ...
Methods are provided for making bispecific antibodies and antibody conjugates comprising site-specifically cross-linking two or more antibodies, antibody fragments or Fc-fusion proteins. Also provided
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Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein
We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that ...
The 3′ major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3′ major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is ...
SIA crosslinker is a non-cleavable, heterobifunctional protein crosslinker. SIA crosslinking reagent is the shortest amine and sulfhydryl (thiol) reactive protein crosslinker.
Using chemical cross-linkers to map contacts among amino acids, structural biologists predict that soluble tau is, in fact, a compact globule containing β-sheets poised to snap into a pathological formation.. ...
Using chemical cross-linkers to map contacts among amino acids, structural biologists predict that soluble tau is, in fact, a compact globule containing β-sheets poised to snap into a pathological formation.. ...
Hello, I am attempting to perform some experiments that inwolve cross-linking with Pierce photoreactive bifunctional cross-linker APDP. As I have no practice using that reagent, any advice would be most welcome. I plan to label one protein via sulphydryl end of APDP and then use the labelled protein in a reaction. After that the glycerol gradient will be run, and appropriate fractions will be flashed with light to activate the photoreactive group of APDP and cross-link that protein to whatever it has in proximity. I have read the papers provided by Pierce, but still have some questions, as follows: - Does APDP decompose thermally readily? Will it be possible to heat APDP-bound protein (that first one) for some 10 min, or will it destroy the photoreactive end (as I am affraid)? And how long can I keep APDP-bound protein sample before actual cross-linking with flashlight? - How photosensitive it is? Do I have to worry about ambient light when labelling the first protein by sulphydryl? - During the ...
Current hydrogels based on chondroitin sulfate (ChS) generally lack the necessary strength and precise mechanical tunability. Addressing these limitations, covalent cross-linking has evolved to produce hydrogels with desirable properties. However, such a methodology always precludes injection and self-healin
The present invention relates to a method of cross-linking a polymeric carbohydrate material with a second material by means of a soluble carbohydrate polymer and a crosslinking agent. The present invention furthermore relates to the resulting cross-linked material, to uses of the cross-linked material, as well as to a kit comprising the soluble carbohydrate polymer and the cross-linking agent. ...
Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents contain two or more reactive ends that are capable of attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other article describes some of the different crosslinkers and their use.
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Probing Akt-Inhibitor Interaction by Chemical Cross-Linking and Mass Spectrometry | Bill X. Huang; Hee-Yong Kim | download | BookSC. Download books for free. Find books
Various cross-linking methods have been developed for labeling of proteins (e.g. IgG) with fluorophores or enzyme (e.g. HRP). Cross-linking is the implementation of covalent bonds between functional groups of amino acids within a protein (intramolecular), or between interacting proteins (intermolecular) using chemical reagents.. Since the SERS-nanotags are indeed several thousand fold heavier than IgG, fluorophores or enzymes, particular attention must be paid to the chosen buffers and bioconjugation technique, which allows the linkage of bio- and nanomaterials [9]. Thus, for bioconjugation of SERS labels, gentle modification and adjustments are required. As it has been shown before, the stability and biofunctionality of SERS nanotags depends on the amount of crosslinker and the avoidance of centrifugation steps [9]. Figure 2C and D demonstrate two different preparations of anti p63 coupled SERS probes with and without the use of a reaction/separation chamber [9, 21]. The p63 IgG labeled SERS ...
Various cross-linking methods have been developed for labeling of proteins (e.g. IgG) with fluorophores or enzyme (e.g. HRP). Cross-linking is the implementation of covalent bonds between functional groups of amino acids within a protein (intramolecular), or between interacting proteins (intermolecular) using chemical reagents.. Since the SERS-nanotags are indeed several thousand fold heavier than IgG, fluorophores or enzymes, particular attention must be paid to the chosen buffers and bioconjugation technique, which allows the linkage of bio- and nanomaterials [9]. Thus, for bioconjugation of SERS labels, gentle modification and adjustments are required. As it has been shown before, the stability and biofunctionality of SERS nanotags depends on the amount of crosslinker and the avoidance of centrifugation steps [9]. Figure 2C and D demonstrate two different preparations of anti p63 coupled SERS probes with and without the use of a reaction/separation chamber [9, 21]. The p63 IgG labeled SERS ...
PRODUCTO ETX Cross-linking agent that improves attachment and wash fastness of resins. PRODUCTO R3 conc. Self-cross-linking agent of resin that improves wash and rubbing fastness of pigments.
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SF3B4 is one of four subunits of the splicing factor 3B. The protein cross-links to a region in the pre-mRNA immediately upstream of the branchpoint…
Tris-EDTA is used to dilute and store nucleic acid samples. Tris-EDTA-based solutions break protein cross-links and can therefore unmask antigens and epitopes in formalin-fixed and paraffin-embedded tissue sections. Treatment with TE Buffer enhances the staining intensity of antibodies in the immuno-histochemical detection of certain proteins.
There is provided herein a membrane or film comprising one or more aromatic ionomers covalently crosslinked through aryl-aryl (--Ar--Ar--), aryl-ether-aryl (--Ar--O--Ar--), aryl-sulfide-aryl (--Ar--S--Ar--), aryl-sulfone-aryl bonds, or any combination thereof, wherein said one or more aromatic ionomers further comprises at least one electron withdrawing group adapted to improve oxidant resistance of said membrane or film.
WESTBURY, NY--(Marketwired - September 11, 2015) - The crosslinking process utilizes short wave (254 nm) UV light to covalently bond nucleic acids to either nitrocellulose or nylon membranes. Spectronics Corporation manufactures advanced, versatile and accurate crosslinkers to avoid the loss of nucleic acids during the hybridization process, a consistent, irritating...
Bio-Synthesis offers peptide conjugation using various cross-linking chemistries. Carrier protein such as KLH, BSA, OVA can be conjugated to peptides.
Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence. J Biol Chem. 1993 Dec 15; 268(35):26745-51 ...
Skin Factor 1 - 40000 - - AGE-breakerSkin Factor 1 is an AGE-breaker. AGE stands fornAdvanced-Glyvcation-Endproducts (AGE) and are crosslinked proteins and sugars. These
1XW7: Diabetes-associated mutations in human insulin: crystal structure and photo-cross-linking studies of a-chain variant insulin wakayama
The Spectrolinker XL-1000 UV crosslinker was made for applications including eliminating PCR contamination, cross-linking DNA an...
Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
When a low concentrations of crosslinker is added to resist (a film used to lay down the patterns of ever-shrinking lines and features on a chip, shown here at left), it is able to pattern smaller ...
TY - JOUR. T1 - Intramolecular crosslinking of hemoglobin A by sulfosuccinimidyl suberate. T2 - Application of crosslinked protein as a blood substitute. AU - Manjula, B. N.. AU - Acharya, A. S.. PY - 1994/11. Y1 - 1994/11. N2 - Sulfosuccinimidyl esters could be targeted to the residues of ββ cleft of hemoglobin A and the bis-sulfosuccinimidyl esters of aliphatic dicarboxylic acids could serve as the `affinity directed ββ crosslinkers of HbA. The reactivity of sulfosuccinimidyl esters of tartaric acid and sebacic acid with HbA was also investigated to establish the appropriate length of the alkyl chain optimal for the crosslinking reaction.. AB - Sulfosuccinimidyl esters could be targeted to the residues of ββ cleft of hemoglobin A and the bis-sulfosuccinimidyl esters of aliphatic dicarboxylic acids could serve as the `affinity directed ββ crosslinkers of HbA. The reactivity of sulfosuccinimidyl esters of tartaric acid and sebacic acid with HbA was also investigated to establish the ...
DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.
Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [3H]BK and [3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacological specificity of the binding of these two radioligands. Cross-linking experiments were done using a series of bifunctional reagents reactive either primarily to amines (homobifunctional) or reactive to amines in one end and to sulfhydryls in the opposite end (heterobifunctional). All the heterobifunctional reagents plus the homobifunctional arylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the [3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 μM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at ≤1 mM, were only able to cross-link ...
For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular ...
ABCA formed a novel intramolecular cross-linking adduct on human serum albumin in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighbouring protein nucleophile. Adducts were detected on lysine, histidine, and cysteine residues in subdomain IB of human serum albumin. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi. Modelling the docking of ABCA with HLA B*57:01 confirmed that ABCA has a strong binding affinity when bound covalently to Ser116, a key residue with regards to recognition by ABC-specific CD8+ T cells. ...
215597-96-9,CCD No.:CCD00211814,Formula:C12 H10 N2 O14 S2 . 2 Na,MolWeight:516.323,Synonyms:BIS(SULFOSUCCINIMIDYL)SUCCINATE SODIUM SALT; Molecular Structure,Chemical Cloud Database
TY - JOUR. T1 - 4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA. AU - Nishimoto, Atsushi. AU - Jitsuzaki, Daichi. AU - Onizuka, Kazumitsu. AU - Taniguchi, Yosuke. AU - Nagatsugi, Fumi. AU - Sasaki, Shigeki. PY - 2013/7. Y1 - 2013/7. N2 - The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson-Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite ...
TY - JOUR. T1 - Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker. AU - Williams, Berea A R. AU - Chaput, John C.. PY - 2010. Y1 - 2010. N2 - Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step.. AB - Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety ...
Sigma-Aldrich offers bifunctional and trifunctional linkers and crosslinking reagents for various conjugation applications. There are numerous bioconjugation possibilities with our linkers and crosslinkers, offering potential for structural stability or assistance in protein-protein, protein-peptide and peptide/protein-small molecule interactions. Other applications include immobilization for assays or purification, as well as various peptide-nucleic acid and nucleic acid-nucleic acid conjugations, among many others. Our homo- and heterofunctional linkers contain diverse functional groups, such as primary amines, sulfhydryls, acids, alcohols and bromides. Many of our crosslinkers are functionalized with maleimide (sulfhydral reactive) and succinimidyl ester (NHS) or isothiocyanate (ITC) groups that react with amines. A selection of mono-protected (Boc, Fmoc, and Cbz) linkers is also available.
Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted ...
The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process cisplatin−DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP[subscript 6], was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSβ, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSβ binding to a cisplatin ICL. ...
Dear netter, I am trying to cross-link some of my mutated actin monomers within filament actin by using the zero-length cross-linker. Anybody has success with this cross-linker? Your protocol and advice will be highly appreciated. Bing ...
TY - JOUR. T1 - Sulfhydryl Site-Specific Cross-Linking and Labeling of Monoclonal Antibodies by a Fluorescent Equilibrium Transfer Alkylation Cross-Link Reagent. AU - del Rosario, Renato B.. AU - Wahl, Richard L.. AU - Brocchini, Stephen J.. AU - Lawton, Richard G.. AU - Smith, Richard H.. PY - 1990/1/1. Y1 - 1990/1/1. N2 - The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of equilibrium transfer alkylation cross-link (ETAC) reagents is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with a,a-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(α,α-bis(p-tolylsulfonylmethyl) acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from lb indicated the formation of S-ETAC-S interchain heavy and ...
Thermo Scientific™ Pierce™ DSS Crosslinker 1g Thermo Scientific™ Pierce™ DSS Crosslinker NHS-ester Crosslinkers
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TY - CONF. T1 - Preparation of porous tubular scaffolds for vascular tissue engineering by photo-crosslinking of PTMC in a glass mold. AU - Guo, Zhengchao. AU - Grijpma, Dirk W.. AU - Poot, Andreas A.. PY - 2015/11/7. Y1 - 2015/11/7. KW - METIS-314723. M3 - Poster. T2 - International Conference on Biofabrication 2015. Y2 - 7 November 2015 through 9 November 2015. ER - ...
Enzymes are immobilized by dissolving in water a photo-crosslinking resin containing stilbazolium groups, vinyl alcohol units and vinyl acetate units, adding an enzyme to the resultant aqueous solution and exposing the enzyme-containing resin solution to light to induce a crosslinking reaction of the photo-crosslinking resin and produce a polymer containing the enzyme entrapped therein.
Shop a large selection of Crosslinking Reagents products and learn more about Thermo Scientific DSS (disuccinimidyl suberate), No-Weigh Format 50mg:Life 50mg.
It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 x g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the
TY - JOUR. T1 - Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8. AU - Wang, Jin. AU - Zhang, Da Wei. AU - Lei, Ying. AU - Xu, Fang. AU - Cohen, Jonathan C.. AU - Hobbs, Helen H.. AU - Xie, Xiao Song. PY - 2008/5/6. Y1 - 2008/5/6. N2 - ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette half-transporters that limit intestinal uptake and promote biliary secretion of neutral sterols. Here, we describe the purification of endogenous G5G8 from mouse liver to near homogeneity. We incorporated the native proteins into membrane vesicles and reconstituted sterol transfer. Native gel electrophoresis, density-gradient ultracentrifugation, and chemical cross-linking studies indicated that the functional native complex is a heterodimer. No higher order oligomeric forms were observed at any stage in the catalytic cycle. Sterol transfer activity by purified native G5G8 was stable, stereospecific, and selective. We also report that G5 but not G8 is S-palmitoylated and that ...
Here we describe a protocol based mostly on structure-guided cysteine cross-linking and proteolysis-coupled gel evaluation to probe conformational modifications of a goal transporter in reside Escherichia coli cells. Although cross-linking approaches have been used to probe the proximity between transmembrane segments in membrane proteins in vivo, to our information this protocol is the primary for use to interrogate transporter dynamics in cells. The use of this protocol is perfect for proteins with recognized or modeled buildings to information the alternative of particular residues with cysteines and the choice of cross-linking brokers with varied spacer arm lengths. This protocol permits for discriminating simply cross-linked and uncross-linked species and doesnt require the customarily troublesome or unavailable reconstitution of transport exercise in an in vitro system. In addition, this protocol could possibly be used to probe the conformation of transporters in cells handled with ...
Summary. Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement ...
Tissue transglutaminase (tTG) belongs to the family of transglutaminase enzymes that catalyze the posttranslational modification of proteins via Ca(2+)-dependent cross-linking reactions. The catalytic action of tTG results in the formation of an isopeptide bond that is of great physiological significance since it is highly resistant to proteolysis and denaturants. Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear. Recent data, however, suggest that the protein polymers resulting from tTG-catalyzed reactions may play a role in commitment of cells to undergo apoptosis. On the same token, tTG-mediated formation of insoluble protein aggregates may underlie the markers of numerous pathological conditions, such as the senile plaques in Alzheimers disease and the Lewy bodies in Parkinsons disease. In addition to catalyzing Ca(2+)-dependent cross-linking reactions, tTG can also
Tissue engineering typically requires a use of scaffolds when delivering tissue-specific cells to be engineered. Hydrogels are frequently used as scaffolds, because their composition, structure, and function resemble the natural tissue extracellular matrix. In this study, hyaluronate-alginate hybrid (HAH) was synthesized by conjugating alginate (ALG) with the hyaluronate (HA) backbone using various types of linkers. HAH hydrogel was prepared by physically cross-linking the HAH polymer in the presence of calcium ions without chemical cross-linkers. The mechanical stiffness of HAH hydrogel was significantly affected by changing the type of a linker between HA and ALG. The mechanical stiffness increased with increasing linker length, likely due to enhanced intermolecular reactions between HA and ALG. This enables controlling the mechanical properties of HAH hydrogels. The types of linkers used to synthesize HAHs also influenced the chondrogenic differentiation of ATDC5 cells cultured in HAH ...
2005 (English)In: 8th International Symposium of Polymers for Advanced Technologies, 13th-16th Sept. 2005, Budapest, Hungary, 2005Conference paper, Published paper (Other scientific) ...
These overt changes in the BM microenvironment most likely limit bloodstream formation in marrow as indicated by patchy hematopoiesis, and even more bloodstream cells are stated in various other organs, like the spleen.69 Lysyl oxidase (LOX) may stabilize collagen fibrils by covalent crosslinking70 and plays a part in solid tumor progression by matrix stiffening.53 Interestingly, megakaryocytes produced from principal myelofibrosis patients present upregulated LOX appearance, facilitating collagen crosslinking thereby.71 While direct ramifications of matrix technicians on the development of principal myelofibrosis remain unidentified, biomaterial strategies, such as for example minimal matrix types of scars68 and interpenetrating polymer systems,54 could be utilized to recapitulate key matrix compositions from the pathological marrow also to differ substrate stiffness independently. V.?BIOPHYSICAL Legislation OF Immune system CELLS WITH IMPLICATIONS IN Cancer tumor IMMUNOTHERAPY Constructed ...
aldehydes chemical defense food quality protein cross-linking protein modifications Weissflog, Jerrit; Adolph, Sven; Wiesemeier, Theresa; Pohnert, Georg
Polyamides comprising substantial amounts of Nylon-11 and/or Nylon-12 units are cross-linked by irradiation in the presence of an unsaturated cross-linking agent, preferably triallyl isocyanurate. The cross-linked products are particularly useful in the form of heat-recoverable shaped articles, e.g. heat-shrinkable tubing.
Various structural components of biological membranes are asymmetrically localized in the two surfaces of the membrane bilayer. This asymmetry is absolute for membrane (glyco) proteins, but only a partial asymmetry has been observed for membrane phospholipids. In the red cell membrane, choline-phospholipids are localized mainly in the outer monolayer whereas aminophospholipids are distributed almost exclusively in the inner monolayer. Several evidences are now available to suggest that this distribution of membrane phospholipids in red cells is directly or indirectly maintained by the membrane-associated cytoskeleton (membrane skeleton). This belief is well supported by the previous as well as recent studies carried out in the authors laboratory. Previously, it has been shown that lipid-lipid interactions play no major role in maintaining the transmembrane phospholipid asymmetry in erythrocytes, and that the asymmetry is lost upon covalent crosslinking of the major membrane skeletal protein, ...
In order for transplanted cells to survive in the body, they need to be gently implanted, as well as provide a suitable environment allowing them to attach, survive, grow and function. Because different cell types prefer different types of environments, the delivery matrix can be modified. The HyStem® hydrogels permit gentle implantation by supporting cells when they are injected through a needle. HyStem® hydrogels are highly customizable, allowing for a variation of gel stiffness, viscosity and gelation time. They are also biocompatible. BioTime has designed these hydrogels to dissolve when needed to gently release cells from the matrix.. In addition to cells, proteins and/or other biological factors it can also be used as a time-release depot for drugs or biological molecules.. The technology underlying BioTimes HyStem® hydrogels was developed at the University of Utah and is based on a unique chemical cross-linking strategy. Building upon this platform, BioTime has developed the HyStem ...
Microcapsules are formed in the absence of coacervation by providing an oil-in-water emulsion containing a polymeric, emulsifying agent having cross-linkable groups or complexing sites and admixing with the emulsion a cross-linking agent or a complexing agent which forms an impermeable coating around the dispersed oil droplets. The emulsifying agent may be non-proteinaceous or the protein, gelatin. Impermeable capsule walls are formed solely by the addition of the cross-linking or complexing agent and extraneous hardening agents are obviated. Moreover, the emulsifying agent may be a preformed, polymeric, cross-linking agent which eliminates the need for any separate cross-linking agent.
A number of clinically important antitumor agents such as cisplatin, cyclophosphamide (a nitrogen mustard) or carmustine (BCNU, a chloro ethyl nitroso urea) for...
556 The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are naturally occurring antitumor antibiotics isolated from various Streptomyces species. They bind covalently in the minor groove of DNA at purine-guanine-purine motifs. SJG-136, a pyrrolobenzodiazepine dimer based on two PBD units joined through their C8-positions via a propyldioxy linker, is presently in Phase I evaluation in both the UK (through Cancer Research UK) and the USA (through the NCI) as a result of its striking in vitro and in vivo activity. As it contains two electrophilic centers, SJG-136 forms interstrand cross-links between guanines on opposite strands of DNA at Purine-GATC-Pyrimidine sequences. More recently we have explored the synthesis and biological activity of C2-aryl substituted PBD monomers that monoalkylate guanines in the minor groove of DNA rather than form cross-links. In this respect, their mechanism of action is similar to Ecteinascidin-743 (Yondelis, ET-743). We have found that these molecules possess encouraging ...
Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent natu
5-(hexyloxy)psoralen: forms a conjugate with oligonucleotide primers (at its omega-hexyl position) to promote site-specific photo-induced cross-linking at the end of the DNA fragment of interest before analysis by DGGE
Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH 2 -terminal targeting presequence that are imported in a linear NH 2 -terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX 3 C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests ...
Crosslinkable compositions contain a calcium carbonate-rich filler coated with a carboxylic acid of the formula R.sup.4--(OCR.sup.3.sub.2--C(.dbd.O)OH).sub.y. The compositions exhibit low modulus, good adhesion to substrates, and low skin formation time.
To understand outcomes, traditional data collection will only take you so far. IQVIA offers methods and expertise that take you to where you want to be. Explore our Innovative data collection techniques applied to a wide range of sources, cross-linking data and closing gaps. Use classical observational, low interventional clinical studies, or novel hybrid approaches such as mosaic and enriched studies. Designed to generate actionable insights and give you validated, representative, and fast results. Discover our track record in EMA-accepted Drug Utilization Studies (DUS) in multiple therapy areas and geographies and our experience in working with the FDA, ENCePP, EUnetHTA , MHRA.. ...
Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
Cross-links are bonds that link one polymer chain to another. These bonds can either be covalent or ionic in nature. In polymer science, the use of cross-links to promote a difference in a polymers physical properties is referred to as cross-linking. When polymer chains are linked together by cross-links, they lose some of their ability to move as individual polymer chains. For example, a liquid polymer, which possesses freely flowing chains can be turned into a solid or gel by cross-linking the chains together. Cross-linking inhibits close packing of the polymer chains, preventing the formation of crystalline regions. The restricted molecular mobility of a cross-linked structure limits the extension of the polymer material under loading. This means that when a polymer is stretched, the cross-links prevent the individual chains from sliding past each other. In the process, the chains may straighten out, but once the stress is removed they return to their original position and the object ...
A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus. Such a domain (LSL 150), recently described as a novel fusion tag, is based on a β-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL 150 to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the ...
Water swellable polyacrylate articles are made from a solution of the polyacrylate having an effective amount of a soluble crosslinking agent therein by heating and/or drying the solution. The polyacrylate solution is made from a polyacrylate by saponification and the cross-linking agent is then added.