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    Name:
    EDC 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride
    Description:
    Thermo Scientific Pierce EDC is a water soluble carbodiimide crosslinker that activates carboxyl groups for spontaneous reaction with primary amines enabling peptide immobilization and hapten carrier protein conjugation Characteristics of EDC • Reactive group carbodiimide • Reaction target activates carboxyl groups to conjugate to amino groups primary amines • Several conjugation strategies react EDC alone with target groups or include NHS or Sulfo NHS to increase reaction efficiency or to stabilize active intermediate for later reaction to amines • Neutral linkage forms neutral amide bonds between carboxyls and amines • Water soluble reagent add directly to reactions in aqueous physiological buffers • Soluble reaction byproducts easily removed by washing with water or dilute acid • High purity crystalline reagent use to create high quality activated derivatives Properties of EDC • Molecular formula C8H17N3 HCl • Molecular weight 191 7 • Spacer arm length 0 Å • CAS Number 25952 53 8 • Reactive groups carbodiimide • Reactivity Forms active intermediate with carboxyl groups at pH 4 7 6 0 optimum then intermediate reacts with primary amines 1 Ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride EDC or EDAC is a zero length crosslinking agent used to couple carboxyl groups to primary amines This crosslinker has been used in diverse applications such as forming amide bonds in peptide synthesis attaching haptens to carrier proteins to form immunogens labeling nucleic acids through 5 phosphate groups and creating amine reactive NHS esters of biomolecules EDC reacts with a carboxyl to form an amine reactive O acylisourea intermediate If this intermediate does not encounter an amine it will hydrolyze and regenerate the carboxyl group In the presence of N hydroxysulfosuccinimide Sulfo NHS EDC can be used to convert carboxyl groups to amine reactive Sulfo NHS esters This is accomplished by mixing the EDC with a carboxyl containing molecule and adding Sulfo NHS Applications • Conjugate carboxyl and amino groups among peptides and proteins • Couple haptens to immunogenic carrier proteins e g attach a peptide to KLH • Immobilize peptide antigens to affinity purify antibodies • Create NHS activated amine reactive labeling compounds • Crosslink proteins to carboxyl coated beads or surfaces • Activate nanoparticles with amine reactive Sulfo NHS esters • DNA labeling through 5 phosphate groups see Tech Tip 30 Product References Crosslinker Application Guide search for recent literature references for this product Related Products Pierce Premium Grade EDC 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride Sulfo NHS N hydroxysulfosuccinimide
    Catalog Number:
    22980
    Price:
    None
    Applications:
    Protein Biology|Protein Crosslinking|Protein Labeling & Crosslinking
    Category:
    Labeling Detection Products
    Buy from Supplier


    Structured Review

    Thermo Fisher edc
    The content of <t>EdU,</t> <t>EdC</t> and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.
    Thermo Scientific Pierce EDC is a water soluble carbodiimide crosslinker that activates carboxyl groups for spontaneous reaction with primary amines enabling peptide immobilization and hapten carrier protein conjugation Characteristics of EDC • Reactive group carbodiimide • Reaction target activates carboxyl groups to conjugate to amino groups primary amines • Several conjugation strategies react EDC alone with target groups or include NHS or Sulfo NHS to increase reaction efficiency or to stabilize active intermediate for later reaction to amines • Neutral linkage forms neutral amide bonds between carboxyls and amines • Water soluble reagent add directly to reactions in aqueous physiological buffers • Soluble reaction byproducts easily removed by washing with water or dilute acid • High purity crystalline reagent use to create high quality activated derivatives Properties of EDC • Molecular formula C8H17N3 HCl • Molecular weight 191 7 • Spacer arm length 0 Å • CAS Number 25952 53 8 • Reactive groups carbodiimide • Reactivity Forms active intermediate with carboxyl groups at pH 4 7 6 0 optimum then intermediate reacts with primary amines 1 Ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride EDC or EDAC is a zero length crosslinking agent used to couple carboxyl groups to primary amines This crosslinker has been used in diverse applications such as forming amide bonds in peptide synthesis attaching haptens to carrier proteins to form immunogens labeling nucleic acids through 5 phosphate groups and creating amine reactive NHS esters of biomolecules EDC reacts with a carboxyl to form an amine reactive O acylisourea intermediate If this intermediate does not encounter an amine it will hydrolyze and regenerate the carboxyl group In the presence of N hydroxysulfosuccinimide Sulfo NHS EDC can be used to convert carboxyl groups to amine reactive Sulfo NHS esters This is accomplished by mixing the EDC with a carboxyl containing molecule and adding Sulfo NHS Applications • Conjugate carboxyl and amino groups among peptides and proteins • Couple haptens to immunogenic carrier proteins e g attach a peptide to KLH • Immobilize peptide antigens to affinity purify antibodies • Create NHS activated amine reactive labeling compounds • Crosslink proteins to carboxyl coated beads or surfaces • Activate nanoparticles with amine reactive Sulfo NHS esters • DNA labeling through 5 phosphate groups see Tech Tip 30 Product References Crosslinker Application Guide search for recent literature references for this product Related Products Pierce Premium Grade EDC 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride Sulfo NHS N hydroxysulfosuccinimide
    https://www.bioz.com/result/edc/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    edc - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine"

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    Journal: Open Biology

    doi: 10.1098/rsob.150172

    The content of EdU, EdC and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.
    Figure Legend Snippet: The content of EdU, EdC and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.

    Techniques Used: Incubation

    The impact of THU and specific siRNAs on the incorporation of EdU into DNA. ( a ) The EdU-derived signal intensity in HeLa cells incubated for 2 h with EdC or EdU in the presence of THU. For the detection of EdU, a click reaction was used. The data are normalized to percentage of the signal of control cells incubated with EdC or EdU without THU (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( b ) The impact of siRNA against CDD and DCTD on the incorporation of EdU into the DNA in cells incubated for 2 h with EdU or EdC. For the detection of EdU, a click reaction was used. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( c ) The amount of CDD and DCTD measured by immunoblots in cells treated with siRNA against CDD and DCTD. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m.
    Figure Legend Snippet: The impact of THU and specific siRNAs on the incorporation of EdU into DNA. ( a ) The EdU-derived signal intensity in HeLa cells incubated for 2 h with EdC or EdU in the presence of THU. For the detection of EdU, a click reaction was used. The data are normalized to percentage of the signal of control cells incubated with EdC or EdU without THU (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( b ) The impact of siRNA against CDD and DCTD on the incorporation of EdU into the DNA in cells incubated for 2 h with EdU or EdC. For the detection of EdU, a click reaction was used. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( c ) The amount of CDD and DCTD measured by immunoblots in cells treated with siRNA against CDD and DCTD. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m.

    Techniques Used: Derivative Assay, Incubation, Western Blot

    The microscopy analysis of EdC conversion to EdU using an antibody reaction. ( a ) Fluorescence detection of EdU by means of an anti-bromodeoxyuridine antibody (clone B44). HeLa cells were incubated with either 10 µM EdU or 10 µM EdC. Then, the detection of EdU (in green) and DNA using DAPI (in blue) was performed. ( b ) The analysis of the reactivity of anti-bromodeoxyuridine antibody (clone B44) using EdU with biotin at the 5′ end and EdC with biotin at the 3′ or 5′ end. The data were normalized to percentage of the signal provided by EdU (equal to 100%). The data are presented as mean ± s.e.m.
    Figure Legend Snippet: The microscopy analysis of EdC conversion to EdU using an antibody reaction. ( a ) Fluorescence detection of EdU by means of an anti-bromodeoxyuridine antibody (clone B44). HeLa cells were incubated with either 10 µM EdU or 10 µM EdC. Then, the detection of EdU (in green) and DNA using DAPI (in blue) was performed. ( b ) The analysis of the reactivity of anti-bromodeoxyuridine antibody (clone B44) using EdU with biotin at the 5′ end and EdC with biotin at the 3′ or 5′ end. The data were normalized to percentage of the signal provided by EdU (equal to 100%). The data are presented as mean ± s.e.m.

    Techniques Used: Microscopy, Fluorescence, Incubation

    Run-on replication assay and hypotonic introduction of EdUTP and EdCTP, EdCDP and EdCMP. ( a ) The detection of EdU and EdC using Alexa Fluor 488 azide in permeabilized HeLa cells (in green). The nuclear DNA was stained by DAPI (in blue). ( b ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdUTP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdUTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( c ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdCMP, EdCDP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdCMP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( d ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide in HeLa cells after the hypotonic introduction of EdUTP or EdCTP with the concurrent introduction of dTTP. The data are normalized to percentage of the signal of EdUTP- or EdCTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m.
    Figure Legend Snippet: Run-on replication assay and hypotonic introduction of EdUTP and EdCTP, EdCDP and EdCMP. ( a ) The detection of EdU and EdC using Alexa Fluor 488 azide in permeabilized HeLa cells (in green). The nuclear DNA was stained by DAPI (in blue). ( b ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdUTP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdUTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( c ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdCMP, EdCDP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdCMP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( d ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide in HeLa cells after the hypotonic introduction of EdUTP or EdCTP with the concurrent introduction of dTTP. The data are normalized to percentage of the signal of EdUTP- or EdCTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m.

    Techniques Used: Staining, Incubation

    EdU- and dT-content ratios and the dependence of EdU incorporation on EdC concentrations. ( a ) The ratio between the content of EdU and dT in isolated DNA after a 24-h incubation with 10 µM EdU or EdC in five cell lines is shown. ( b ) The average nuclear signal in five cell lines incubated with 0.016–250 µM EdC for 4 h. The detection of the signal was performed using a click reaction. The data were normalized to the signal provided by 250 µM EdC (equal to 100%). The data are presented as mean ± s.e.m.
    Figure Legend Snippet: EdU- and dT-content ratios and the dependence of EdU incorporation on EdC concentrations. ( a ) The ratio between the content of EdU and dT in isolated DNA after a 24-h incubation with 10 µM EdU or EdC in five cell lines is shown. ( b ) The average nuclear signal in five cell lines incubated with 0.016–250 µM EdC for 4 h. The detection of the signal was performed using a click reaction. The data were normalized to the signal provided by 250 µM EdC (equal to 100%). The data are presented as mean ± s.e.m.

    Techniques Used: Isolation, Incubation

    2) Product Images from "Sensing soluble uric acid by Naip1-Nlrp3 platform"

    Article Title: Sensing soluble uric acid by Naip1-Nlrp3 platform

    Journal: bioRxiv

    doi: 10.1101/2020.05.15.077644

    QCM monitoring and SPR sensorgram evidencing all steps involved for the detection of the interaction between sUA and Naip1 protein. (A) QCM responses over time within sUA injection after Naip1 immobilization upon anti-GFP adsorption on the gold quartz crystals surface at 37°C. The arrows indicate sample injection. (B) Schematic representation of the constructed SPR sensor chip (in the box). Sequential addition of compounds into the system: (i) addition of the buffer solution (PBS, 10 mmol L −1 at pH 7.4); (ii) mixture consisting of EDC (150 mmol L −1 ) and NHS (150 mmol L −1 ); (iii) PBS; (iv) immobilization of anti-GFP (10 μg mL −1 ); (v) PBS; (vi) addition of ethanolamine (EA); addition of cell lysates containing Naip1 protein (2 μg mol L −1 ). It is possible observe a very intensive response for the interaction of the Naip1 protein with anti-GFP; (v) PBS;(vi) addition of pure H 2 O; (vii) addition of sUA (2 μmol L −1 , purple line) and palmitate (2 μmol L −1 , green line). It is possible to observe the significant variation of the SPR angle (Δθ SPR ) due to the interaction between sUA and Naip protein. In A and B, data are representative of three independent experiments.
    Figure Legend Snippet: QCM monitoring and SPR sensorgram evidencing all steps involved for the detection of the interaction between sUA and Naip1 protein. (A) QCM responses over time within sUA injection after Naip1 immobilization upon anti-GFP adsorption on the gold quartz crystals surface at 37°C. The arrows indicate sample injection. (B) Schematic representation of the constructed SPR sensor chip (in the box). Sequential addition of compounds into the system: (i) addition of the buffer solution (PBS, 10 mmol L −1 at pH 7.4); (ii) mixture consisting of EDC (150 mmol L −1 ) and NHS (150 mmol L −1 ); (iii) PBS; (iv) immobilization of anti-GFP (10 μg mL −1 ); (v) PBS; (vi) addition of ethanolamine (EA); addition of cell lysates containing Naip1 protein (2 μg mol L −1 ). It is possible observe a very intensive response for the interaction of the Naip1 protein with anti-GFP; (v) PBS;(vi) addition of pure H 2 O; (vii) addition of sUA (2 μmol L −1 , purple line) and palmitate (2 μmol L −1 , green line). It is possible to observe the significant variation of the SPR angle (Δθ SPR ) due to the interaction between sUA and Naip protein. In A and B, data are representative of three independent experiments.

    Techniques Used: SPR Assay, Injection, Adsorption, Construct, Chromatin Immunoprecipitation

    3) Product Images from "Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors"

    Article Title: Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors

    Journal:

    doi: 10.1210/me.2008-0059

    Inhibitors of c-Src Kinase or MEK Dampen E2- and EDC-Induced Gene Regulation
    Figure Legend Snippet: Inhibitors of c-Src Kinase or MEK Dampen E2- and EDC-Induced Gene Regulation

    Techniques Used:

    Time Course of Regulation of Gene Expression by E2 or EDC
    Figure Legend Snippet: Time Course of Regulation of Gene Expression by E2 or EDC

    Techniques Used: Expressing

    EDC and E2 Increase Gene Expression by Increasing Gene Transcription and Not Altering mRNA Stability
    Figure Legend Snippet: EDC and E2 Increase Gene Expression by Increasing Gene Transcription and Not Altering mRNA Stability

    Techniques Used: Expressing

    EDC Is Ineffective in Recruiting ERα to ER Binding Sites of Estrogen Target Genes whereas E2 Elicits a Robust ERα Recruitment
    Figure Legend Snippet: EDC Is Ineffective in Recruiting ERα to ER Binding Sites of Estrogen Target Genes whereas E2 Elicits a Robust ERα Recruitment

    Techniques Used: Binding Assay

    cDNA Microarray Analysis of Genes Regulated by E2 and EDC
    Figure Legend Snippet: cDNA Microarray Analysis of Genes Regulated by E2 and EDC

    Techniques Used: Microarray

    4) Product Images from "Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo"

    Article Title: Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo

    Journal: Molecular pharmaceutics

    doi: 10.1021/acs.molpharmaceut.5b00054

    Schematic Illustration of “Post-Fabrication” siRNA Loading and Surface Modification: (a) NHS-PEG 3.4k -COOH, DMF, Pyridine; (b) SPDP, PBS/CH 3 CN; (c) siRNA-SH, PBS; (d) Poly-L-lysine, EDC, Sulfo-NHS, PBS
    Figure Legend Snippet: Schematic Illustration of “Post-Fabrication” siRNA Loading and Surface Modification: (a) NHS-PEG 3.4k -COOH, DMF, Pyridine; (b) SPDP, PBS/CH 3 CN; (c) siRNA-SH, PBS; (d) Poly-L-lysine, EDC, Sulfo-NHS, PBS

    Techniques Used: Modification

    5) Product Images from "Quantifying and engineering mucus adhesion of probiotics"

    Article Title: Quantifying and engineering mucus adhesion of probiotics

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.9b00356

    Schematic of mucus immobilization strategy The structure of mucus presents an opportunity for covalent immobilization chemistry. Gel-forming mucins such as Muc2 form (a.) macromolecular sheets of trimers of dimers that crosslink or interact to form a mesh between von Willebrand Factor (vWF) domains and cysteine knots (CK). Each mucin monomer has a highly glycosylated proline-threonine/serine (PTS) domain with the (b.) highly sialylated core 3 O-glycan dominating in the intestines. Sialic acid, or N-acetylneuraminic acid (purple diamond), provides a freely available carboxylic acid that can be (c.) coupled to a primary amine presented on a glass or plastic surface using EDC/NHS crosslinking chemistry. Other common glycans include N -acetylglucosamine (blue square), N -acetylgalactosamine (yellow square), galactose (yellow circle), and fucose (red triangle). *Final optimized conditions for full mucus coverage require 1% PIM, 10x higher than other conditions, and ELISA luminescence values increased proportionally.
    Figure Legend Snippet: Schematic of mucus immobilization strategy The structure of mucus presents an opportunity for covalent immobilization chemistry. Gel-forming mucins such as Muc2 form (a.) macromolecular sheets of trimers of dimers that crosslink or interact to form a mesh between von Willebrand Factor (vWF) domains and cysteine knots (CK). Each mucin monomer has a highly glycosylated proline-threonine/serine (PTS) domain with the (b.) highly sialylated core 3 O-glycan dominating in the intestines. Sialic acid, or N-acetylneuraminic acid (purple diamond), provides a freely available carboxylic acid that can be (c.) coupled to a primary amine presented on a glass or plastic surface using EDC/NHS crosslinking chemistry. Other common glycans include N -acetylglucosamine (blue square), N -acetylgalactosamine (yellow square), galactose (yellow circle), and fucose (red triangle). *Final optimized conditions for full mucus coverage require 1% PIM, 10x higher than other conditions, and ELISA luminescence values increased proportionally.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    6) Product Images from "A Missense Mutation in a Highly Conserved Alternate Exon of Dynamin-1 Causes Epilepsy in Fitful Mice"

    Article Title: A Missense Mutation in a Highly Conserved Alternate Exon of Dynamin-1 Causes Epilepsy in Fitful Mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001046

    DNM1a Ftfl is defective in higher order homo-oligomerization. (A) Left panel , protein extract from P14 whole brain tissue of homozygous fitful and wildtype littermates incubated with 0 or 20mM EDC cross-linker and hybridized with anti-dynamin-1 antibody. Monomers migrate at 100kD, dimers at 200kD and the tetramers are at 400kD. This assay was performed over three separate times with different samples each time; a representative blot with corresponding percentages is shown. Mean densities (± 1SD) from all experiments are: wildtype 28.75±8.24 (monomer), 29.67±13.9 (dimer), 43.9±8.5 (tetramer); mutant 44±10.7 (monomer), 39.5±12.2 (dimer), 23±13.5 (tetramer) Right panel , COS-7 cells transfected with DNM1-GFP constructs show differences in dimerization. (B) COS-7 cells doubly transfected with DNM1-GFP and DNM1-HA constructs show isoform heterodimerization. Protein extracts from cells were incubated with 0 or 20mM EDC and analyzed by Western blot. Blots were hybridized with anti-GFP antibody, stripped of antibody and then re-hybridized with anti-HA antibody in order to ascertain the presence of each construct in the dimers. A representative blot hybridized with anti-GFP antibody is shown.
    Figure Legend Snippet: DNM1a Ftfl is defective in higher order homo-oligomerization. (A) Left panel , protein extract from P14 whole brain tissue of homozygous fitful and wildtype littermates incubated with 0 or 20mM EDC cross-linker and hybridized with anti-dynamin-1 antibody. Monomers migrate at 100kD, dimers at 200kD and the tetramers are at 400kD. This assay was performed over three separate times with different samples each time; a representative blot with corresponding percentages is shown. Mean densities (± 1SD) from all experiments are: wildtype 28.75±8.24 (monomer), 29.67±13.9 (dimer), 43.9±8.5 (tetramer); mutant 44±10.7 (monomer), 39.5±12.2 (dimer), 23±13.5 (tetramer) Right panel , COS-7 cells transfected with DNM1-GFP constructs show differences in dimerization. (B) COS-7 cells doubly transfected with DNM1-GFP and DNM1-HA constructs show isoform heterodimerization. Protein extracts from cells were incubated with 0 or 20mM EDC and analyzed by Western blot. Blots were hybridized with anti-GFP antibody, stripped of antibody and then re-hybridized with anti-HA antibody in order to ascertain the presence of each construct in the dimers. A representative blot hybridized with anti-GFP antibody is shown.

    Techniques Used: Incubation, Mutagenesis, Transfection, Construct, Western Blot

    7) Product Images from "Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge"

    Article Title: Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.4161/hv.27567

    Figure 4. Physical characterization of the EDC/NHS TMV-HA conjugate. ( A ) Increased EDC/NHS conjugation time from 30 to 60 min caused additional high molecular weight bands to appear in the conjugated reactions (*), in addition to more accumulation in the well (arrow; T-H reactions). ( B ) Laser Optical scattering microscopy was used to determine the size of the vaccine particle in suspension. Average rotational size was measured for both TMV and TMV-HA, in triplicate. ( C ) 40 000× Electron microscopy imaging of TMV or the TMV-HA conjugate. Particle average diameter increased by 34% from 18.0 nM to 24.1 nM after HA conjugation. The diameter was derived as the average of 30 electron micrograph images, using the bar = 100 nM as a size standard. ( D ) TMV and HA were assessed independently at 100 µg, and the TMV-HA conjugate was assessed at 20 µg for the ability to agglutinate turkey RBCs. Data are shown as the inverse of the minimum titer required to stimulate agglutination.
    Figure Legend Snippet: Figure 4. Physical characterization of the EDC/NHS TMV-HA conjugate. ( A ) Increased EDC/NHS conjugation time from 30 to 60 min caused additional high molecular weight bands to appear in the conjugated reactions (*), in addition to more accumulation in the well (arrow; T-H reactions). ( B ) Laser Optical scattering microscopy was used to determine the size of the vaccine particle in suspension. Average rotational size was measured for both TMV and TMV-HA, in triplicate. ( C ) 40 000× Electron microscopy imaging of TMV or the TMV-HA conjugate. Particle average diameter increased by 34% from 18.0 nM to 24.1 nM after HA conjugation. The diameter was derived as the average of 30 electron micrograph images, using the bar = 100 nM as a size standard. ( D ) TMV and HA were assessed independently at 100 µg, and the TMV-HA conjugate was assessed at 20 µg for the ability to agglutinate turkey RBCs. Data are shown as the inverse of the minimum titer required to stimulate agglutination.

    Techniques Used: Conjugation Assay, Molecular Weight, Microscopy, Electron Microscopy, Imaging, Derivative Assay, Agglutination

    Figure 2. Conjugation of TMV to HA protein. Intact TMV 1295.10 was conjugated to HA protein in vitro as described, and analyzed on an SDS PAGE after staining with Coomassie Blue. ( A ) Glutaraldehyde, ( B ) EDC/Sulfo-NHS or ( C ) SMCC chemistries was successful in conjugating the HA protein to KLH (KLH-HA), TMV (TMV-HA) or as a self-conjugate (HA-HA). The mixture, as indicated by K+H or T+H, sampled before addition of the conjugation chemistry, shows an equimolar representation of both the proteins. The conjugate appears as a high molecular weight aggregate > 190 kDa and in the gel stack. Successful conjugation was qualified by the complete absence of ‘free’ (unbound) HA protein as indicated. K-H (KLH-HA Conjugate), T-H (TMV-HA Conjugate), H-H (HA Self Conjugate).
    Figure Legend Snippet: Figure 2. Conjugation of TMV to HA protein. Intact TMV 1295.10 was conjugated to HA protein in vitro as described, and analyzed on an SDS PAGE after staining with Coomassie Blue. ( A ) Glutaraldehyde, ( B ) EDC/Sulfo-NHS or ( C ) SMCC chemistries was successful in conjugating the HA protein to KLH (KLH-HA), TMV (TMV-HA) or as a self-conjugate (HA-HA). The mixture, as indicated by K+H or T+H, sampled before addition of the conjugation chemistry, shows an equimolar representation of both the proteins. The conjugate appears as a high molecular weight aggregate > 190 kDa and in the gel stack. Successful conjugation was qualified by the complete absence of ‘free’ (unbound) HA protein as indicated. K-H (KLH-HA Conjugate), T-H (TMV-HA Conjugate), H-H (HA Self Conjugate).

    Techniques Used: Conjugation Assay, In Vitro, SDS Page, Staining, Molecular Weight

    8) Product Images from "The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro *"

    Article Title: The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.564740

    EDC cross-linking of Rh-SMM filaments. A , image of Coomassie-stained 3–8% Tris-acetate gel (Invitrogen) showing HiMark prestained high molecular weight protein standards ( Stds ; Invitrogen), 5 μg of Rh-SMM (No XL), and 5 μg of XL-Rh-SMM
    Figure Legend Snippet: EDC cross-linking of Rh-SMM filaments. A , image of Coomassie-stained 3–8% Tris-acetate gel (Invitrogen) showing HiMark prestained high molecular weight protein standards ( Stds ; Invitrogen), 5 μg of Rh-SMM (No XL), and 5 μg of XL-Rh-SMM

    Techniques Used: Staining, Molecular Weight

    9) Product Images from "Identification of a Misfolded Region in Superoxide Dismutase 1 That Is Exposed in Amyotrophic Lateral Sclerosis"

    Article Title: Identification of a Misfolded Region in Superoxide Dismutase 1 That Is Exposed in Amyotrophic Lateral Sclerosis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.581801

    C4F6 specifically cross-links to misfolded SOD1 in the presence of DSP or EDC. A , a nonreducing Western analysis with pan-SOD1 ( red ) and anti-Fab ( green ) antibodies demonstrates the specific cross-linking with DSP between misfolded SOD1 variants and C4F6
    Figure Legend Snippet: C4F6 specifically cross-links to misfolded SOD1 in the presence of DSP or EDC. A , a nonreducing Western analysis with pan-SOD1 ( red ) and anti-Fab ( green ) antibodies demonstrates the specific cross-linking with DSP between misfolded SOD1 variants and C4F6

    Techniques Used: Western Blot

    10) Product Images from "Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification"

    Article Title: Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2009.06.020

    Reaction for the coupling of the carboxylate groups of the protein with an amine in the presence of EDC and HOBt.
    Figure Legend Snippet: Reaction for the coupling of the carboxylate groups of the protein with an amine in the presence of EDC and HOBt.

    Techniques Used:

    11) Product Images from "Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **"

    Article Title: Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **

    Journal: Advanced functional materials

    doi: 10.1002/adfm.201203319

    Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.
    Figure Legend Snippet: Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.

    Techniques Used: Imaging, Fluorescence

    12) Product Images from "CheZ Has No Effect on Flagellar Motors Activated by CheY13DK106YW"

    Article Title: CheZ Has No Effect on Flagellar Motors Activated by CheY13DK106YW

    Journal: Journal of Bacteriology

    doi:

    Immunoblot of cross-linked products of CheZ recorded on film by enhanced chemiluminescence. The molecular mass scale is shown on the right. Symbols on the bottom: +, present; −, absent; wt, wild-type CheY; ∗∗, CheY**; AcP, acetylphosphate (which phosphorylates wild-type CheY); EDC-NHS, cross-linking reagents (see Materials and Methods). Each lane contained 100 ng of CheZ.
    Figure Legend Snippet: Immunoblot of cross-linked products of CheZ recorded on film by enhanced chemiluminescence. The molecular mass scale is shown on the right. Symbols on the bottom: +, present; −, absent; wt, wild-type CheY; ∗∗, CheY**; AcP, acetylphosphate (which phosphorylates wild-type CheY); EDC-NHS, cross-linking reagents (see Materials and Methods). Each lane contained 100 ng of CheZ.

    Techniques Used:

    13) Product Images from "C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization"

    Article Title: C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization

    Journal: Journal of Biomolecular Techniques : JBT

    doi: 10.7171/jbt.13-2401-003

    Protein/peptide glycinamidation. β-Lactoglobulin (50 pmoles) was reduced, carboxyamidomethylated, and glycinamidated in the sequential one-pot reaction sequence. Carbamidation was carried out in a 0.4-M nucleophile, 0.1 M EDC, and 5 mM sulfo-NHS
    Figure Legend Snippet: Protein/peptide glycinamidation. β-Lactoglobulin (50 pmoles) was reduced, carboxyamidomethylated, and glycinamidated in the sequential one-pot reaction sequence. Carbamidation was carried out in a 0.4-M nucleophile, 0.1 M EDC, and 5 mM sulfo-NHS

    Techniques Used: Sequencing

    14) Product Images from "Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface"

    Article Title: Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0711919105

    EDC/sulfo-NHS cross-linking of the acidic C-terminal tail of gp2.5. ( A ) A peptide (80 μM) corresponding to the C-terminal 26 amino acids (residues 206–232) of gp2.5 was activated with EDC/sulfo-NHS. A control peptide (160 μM) containing a random sequence of the same residues also was activated with the same reagent. Each activated peptide was then incubated with 4 μM wild-type gp2.5 for 20 min at room temperature. The cross-linking reaction was stopped with SDS-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad). ( B ) EDC/sulfo-NHS-activated wild-type (80 μM) or random (80, 160, and 320 μM) peptide was cross-linked to gp2.5Δ26 protein (4 μM) for 20 min at room temperature. The cross-linking reaction was stopped with SDS/PAGE-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad).
    Figure Legend Snippet: EDC/sulfo-NHS cross-linking of the acidic C-terminal tail of gp2.5. ( A ) A peptide (80 μM) corresponding to the C-terminal 26 amino acids (residues 206–232) of gp2.5 was activated with EDC/sulfo-NHS. A control peptide (160 μM) containing a random sequence of the same residues also was activated with the same reagent. Each activated peptide was then incubated with 4 μM wild-type gp2.5 for 20 min at room temperature. The cross-linking reaction was stopped with SDS-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad). ( B ) EDC/sulfo-NHS-activated wild-type (80 μM) or random (80, 160, and 320 μM) peptide was cross-linked to gp2.5Δ26 protein (4 μM) for 20 min at room temperature. The cross-linking reaction was stopped with SDS/PAGE-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad).

    Techniques Used: Sequencing, Incubation, SDS Page

    ssDNA-binding and peptide cross-linking are mutually exclusive. ( A ) Gp2.5Δ26 (4 μM) was preincubated with increasing amounts (56–1,120 μM in nucleotides) of Φ X174 circular ssDNA for 10 min on ice. EDC/sulfo-NHS-activated peptide (80 μM) was added, and the reaction was incubated at room temperature for 20 min. The cross-linking reactions were stopped with SDS/PAGE-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad). ( B ) Wild-type C-terminal peptide (80 μM) was activated with EDC/sulfo-NHS and cross-linked to 4 μM gp2.5Δ26. One half of the cross-linking reaction was loaded on an ssDNA spin column, subjected to two low-salt buffer washes, and eluted with high-salt buffer as described in Materials and Methods . Half of the cross-linking reaction was loaded on gel as a reference for the species subjected to ssDNA cellulose binding (lane 1).
    Figure Legend Snippet: ssDNA-binding and peptide cross-linking are mutually exclusive. ( A ) Gp2.5Δ26 (4 μM) was preincubated with increasing amounts (56–1,120 μM in nucleotides) of Φ X174 circular ssDNA for 10 min on ice. EDC/sulfo-NHS-activated peptide (80 μM) was added, and the reaction was incubated at room temperature for 20 min. The cross-linking reactions were stopped with SDS/PAGE-loading buffer, and the products were resolved on 4–20% SDS/PAGE (Bio-Rad). ( B ) Wild-type C-terminal peptide (80 μM) was activated with EDC/sulfo-NHS and cross-linked to 4 μM gp2.5Δ26. One half of the cross-linking reaction was loaded on an ssDNA spin column, subjected to two low-salt buffer washes, and eluted with high-salt buffer as described in Materials and Methods . Half of the cross-linking reaction was loaded on gel as a reference for the species subjected to ssDNA cellulose binding (lane 1).

    Techniques Used: Binding Assay, Incubation, SDS Page

    15) Product Images from "GDNF promotes tubulogenesis of GFR?1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase"

    Article Title: GDNF promotes tubulogenesis of GFR?1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200212174

    GFRα1 does not complex with Met. Binding of 125 I-GDNF to COS7 cells transfected with gfr α 1 and 125 I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125 I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125 I-HGF α subunit and proHGF are marked by arrows. 125 I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.
    Figure Legend Snippet: GFRα1 does not complex with Met. Binding of 125 I-GDNF to COS7 cells transfected with gfr α 1 and 125 I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125 I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125 I-HGF α subunit and proHGF are marked by arrows. 125 I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.

    Techniques Used: Binding Assay, Transfection, SDS Page

    16) Product Images from "A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts"

    Article Title: A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts

    Journal: mBio

    doi: 10.1128/mBio.00745-17

    Effect of ICP22 on the localization of SSRP1 in HSV-1-infected cells. Vero cells were infected with wild-type (KOS) or ICP22 mutant (n199) virus at an MOI of 10. (A) EdC (10 μM) was added to the medium of infected cells from 4 to 6 hpi. The cells were then fixed, stained with Hoechst stain (blue), and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunofluorescence (red). Single cells shown are representative of cells in two independent replicates. (B) Infected cells were fixed at 3 hpi and stained with Hoechst stain (blue). ICP4 (green) and SSRP1 (red) were detected by immunofluorescence. Single cells shown are representative of cells in two independent replicates.
    Figure Legend Snippet: Effect of ICP22 on the localization of SSRP1 in HSV-1-infected cells. Vero cells were infected with wild-type (KOS) or ICP22 mutant (n199) virus at an MOI of 10. (A) EdC (10 μM) was added to the medium of infected cells from 4 to 6 hpi. The cells were then fixed, stained with Hoechst stain (blue), and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunofluorescence (red). Single cells shown are representative of cells in two independent replicates. (B) Infected cells were fixed at 3 hpi and stained with Hoechst stain (blue). ICP4 (green) and SSRP1 (red) were detected by immunofluorescence. Single cells shown are representative of cells in two independent replicates.

    Techniques Used: Infection, Mutagenesis, Staining, Immunofluorescence

    Abundance of FACT complex proteins and localization of SSRP1 in infected cells. (A) MRC5 cells were infected with wild-type (KOS) virus at an MOI of 10. Cell lysates were collected at the indicated hours postinfection. Cell lysates were run on a 10% SDS-PAGE gel and probed with antibodies against Spt16, SSRP1, ICP4, and GAPDH. (B) Vero cells were infected with unlabeled KOS, or KOS prelabeled with 10 μM EdC, at an MOI of 10. Cells infected with prelabeled KOS were fixed at 3 hpi. Cells infected with unlabeled KOS were incubated in medium containing 10 μM EdC starting at 4 hpi and then fixed at 6 hpi. Fixed cells were stained with Hoechst stain (blue) and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunofluorescence (red).
    Figure Legend Snippet: Abundance of FACT complex proteins and localization of SSRP1 in infected cells. (A) MRC5 cells were infected with wild-type (KOS) virus at an MOI of 10. Cell lysates were collected at the indicated hours postinfection. Cell lysates were run on a 10% SDS-PAGE gel and probed with antibodies against Spt16, SSRP1, ICP4, and GAPDH. (B) Vero cells were infected with unlabeled KOS, or KOS prelabeled with 10 μM EdC, at an MOI of 10. Cells infected with prelabeled KOS were fixed at 3 hpi. Cells infected with unlabeled KOS were incubated in medium containing 10 μM EdC starting at 4 hpi and then fixed at 6 hpi. Fixed cells were stained with Hoechst stain (blue) and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunofluorescence (red).

    Techniques Used: Infection, SDS Page, Incubation, Staining, Immunofluorescence

    17) Product Images from "Quantifying and engineering mucus adhesion of probiotics"

    Article Title: Quantifying and engineering mucus adhesion of probiotics

    Journal: bioRxiv

    doi: 10.1101/731505

    Schematic of mucus immobilization strategy The structure of mucus presents an opportunity for covalent immobilization chemistry. Gel-forming mucins such as Muc2 form (a.) macromolecular sheets of trimers of dimers that crosslink or interact to form a mesh between von Willebrand Factor (vWF) domains and cysteine knots (CK). Each mucin monomer has a highly glycosylated proline-threonine/serine (PTS) domain with the (b.) highly sialylated core 3 O-glycan dominating in the intestines. Sialic acid, or N-acetylneuraminic acid (purple diamond), provides a freely available carboxylic acid that can be (c.) coupled to a primary amine presented on a glass or plastic surface using EDC/NHS crosslinking chemistry. Other common glycans include N -acetylglucosamine (blue square), N -acetylgalactosamine (yellow square), galactose (yellow circle), and fucose (red triangle). *Final optimized conditions for full mucus coverage require 1% PIM, 10x higher than other conditions, and ELISA luminescence values increased proportionally.
    Figure Legend Snippet: Schematic of mucus immobilization strategy The structure of mucus presents an opportunity for covalent immobilization chemistry. Gel-forming mucins such as Muc2 form (a.) macromolecular sheets of trimers of dimers that crosslink or interact to form a mesh between von Willebrand Factor (vWF) domains and cysteine knots (CK). Each mucin monomer has a highly glycosylated proline-threonine/serine (PTS) domain with the (b.) highly sialylated core 3 O-glycan dominating in the intestines. Sialic acid, or N-acetylneuraminic acid (purple diamond), provides a freely available carboxylic acid that can be (c.) coupled to a primary amine presented on a glass or plastic surface using EDC/NHS crosslinking chemistry. Other common glycans include N -acetylglucosamine (blue square), N -acetylgalactosamine (yellow square), galactose (yellow circle), and fucose (red triangle). *Final optimized conditions for full mucus coverage require 1% PIM, 10x higher than other conditions, and ELISA luminescence values increased proportionally.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    18) Product Images from "HSPB7 is indispensable for heart development by modulating actin filament assembly"

    Article Title: HSPB7 is indispensable for heart development by modulating actin filament assembly

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1713763114

    HSPB7 binds G actin and inhibits actin polymerization. ( A ) InstantBlue-stained SDS/PAGE gel image of positive control [α-actinin (Actn2)] and negative control (BSA) for F-actin cosedimentation binding assay. ( B ) InstantBlue-stained SDS/PAGE gel image of G-actin and F-actin preparation for blot overlay assay after ultracentrifugation. Note that there was a small amount of unpolymerized G actin still present in F-actin preparation as well as a small amount of polymerized F actin present in G-actin preparation. P, pellet; S, supernatant; T, total actin. ( C ) Western blot images of blot overlay assay using an antibody against actin ( Upper ). HSPB7, αB-crystallin, Actn2 (positive control), and BSA (negative control) proteins were immobilized on Nitrocellulose membrane and incubated with G actin for 1 h at room temperature; then, the membrane was washed and immunoblotted with antibody against actin to reveal potential interactions between test proteins and G actin. The presence of HSPB7 or αB-crystallin on the Nitrocellulose membrane was confirmed by immunoblots using antibody raised against either HSPB7 or αB-crystallin ( Lower ). IB, immunoblot. ( D ) Silver stain and Western blot images of HSPB7 cross-linking with G actin by EDC/NHS. *HSPB7–actin complex; # band with molecular size consistent with a complex containing actin and HSPB7 degradation products (∼13 kDa) found during HSPB7 purification.
    Figure Legend Snippet: HSPB7 binds G actin and inhibits actin polymerization. ( A ) InstantBlue-stained SDS/PAGE gel image of positive control [α-actinin (Actn2)] and negative control (BSA) for F-actin cosedimentation binding assay. ( B ) InstantBlue-stained SDS/PAGE gel image of G-actin and F-actin preparation for blot overlay assay after ultracentrifugation. Note that there was a small amount of unpolymerized G actin still present in F-actin preparation as well as a small amount of polymerized F actin present in G-actin preparation. P, pellet; S, supernatant; T, total actin. ( C ) Western blot images of blot overlay assay using an antibody against actin ( Upper ). HSPB7, αB-crystallin, Actn2 (positive control), and BSA (negative control) proteins were immobilized on Nitrocellulose membrane and incubated with G actin for 1 h at room temperature; then, the membrane was washed and immunoblotted with antibody against actin to reveal potential interactions between test proteins and G actin. The presence of HSPB7 or αB-crystallin on the Nitrocellulose membrane was confirmed by immunoblots using antibody raised against either HSPB7 or αB-crystallin ( Lower ). IB, immunoblot. ( D ) Silver stain and Western blot images of HSPB7 cross-linking with G actin by EDC/NHS. *HSPB7–actin complex; # band with molecular size consistent with a complex containing actin and HSPB7 degradation products (∼13 kDa) found during HSPB7 purification.

    Techniques Used: Staining, SDS Page, Positive Control, Negative Control, Binding Assay, Overlay Assay, Western Blot, Incubation, Silver Staining, Purification

    19) Product Images from "Inner lumen proteins stabilize doublet microtubules in cilia and flagella"

    Article Title: Inner lumen proteins stabilize doublet microtubules in cilia and flagella

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09051-x

    Characteristics of the FAP45 and FAP52 null mutants. a Top: a transverse view of the 9+2 structure of the Chlamydomonas axoneme. Scale bar = 50 nm. Bottom: a magnified DMT. Arrowheads indicate MIPs. Major structures are colored; Outer dynein arm (ODA, magenta); Inner dynein arm (IDA, green); Dynein regulatory complex (DRC, purple); Radial spoke (RS, blue). IJ inner junction, OJ outer junction. Scale bar = 25 nm. b Western blot analyses of wild type, fap45 , fap52 , and fap45fap52 double mutant axonemes stained with various antibodies. FAP45 and FAP52 proteins were not detected in fap45 and fap52 , respectively. Proteins essential for flagellar motility (ODA-IC2: outer dynein arm-intermediate chain 2; IDA-IC140: inner dynein arm-intermediate chain 140; IDA-p28: inner dynein arm-light chain p28; RSP1: radial spoke protein 1; DRC2 and 4: dynein regulatory complex 2 and 4; FAP20: inner junction protein of DMT) were not reduced in the mutants. c , d Axonemes from wild type and mutant Chlamydomonas crosslinked using EDC (zero-length crosslinker) were immunoblotted with anti-FAP45 ( c ) and anti-FAP52 antibodies ( d ). Filled arrowheads indicate the crosslinked product of FAP45 and FAP52 in a 1:1 ratio. The open arrowhead indicates the crosslinked product of FAP45 and tubulin. e – g Motility phenotypes of the mutants were assayed using the CLONA system. Swimming velocity and beat frequency were slightly reduced in fap45 , whereas no significant reduction was observed in fap52 . fap45fap52 showed a more severe phenotype than did fap45
    Figure Legend Snippet: Characteristics of the FAP45 and FAP52 null mutants. a Top: a transverse view of the 9+2 structure of the Chlamydomonas axoneme. Scale bar = 50 nm. Bottom: a magnified DMT. Arrowheads indicate MIPs. Major structures are colored; Outer dynein arm (ODA, magenta); Inner dynein arm (IDA, green); Dynein regulatory complex (DRC, purple); Radial spoke (RS, blue). IJ inner junction, OJ outer junction. Scale bar = 25 nm. b Western blot analyses of wild type, fap45 , fap52 , and fap45fap52 double mutant axonemes stained with various antibodies. FAP45 and FAP52 proteins were not detected in fap45 and fap52 , respectively. Proteins essential for flagellar motility (ODA-IC2: outer dynein arm-intermediate chain 2; IDA-IC140: inner dynein arm-intermediate chain 140; IDA-p28: inner dynein arm-light chain p28; RSP1: radial spoke protein 1; DRC2 and 4: dynein regulatory complex 2 and 4; FAP20: inner junction protein of DMT) were not reduced in the mutants. c , d Axonemes from wild type and mutant Chlamydomonas crosslinked using EDC (zero-length crosslinker) were immunoblotted with anti-FAP45 ( c ) and anti-FAP52 antibodies ( d ). Filled arrowheads indicate the crosslinked product of FAP45 and FAP52 in a 1:1 ratio. The open arrowhead indicates the crosslinked product of FAP45 and tubulin. e – g Motility phenotypes of the mutants were assayed using the CLONA system. Swimming velocity and beat frequency were slightly reduced in fap45 , whereas no significant reduction was observed in fap52 . fap45fap52 showed a more severe phenotype than did fap45

    Techniques Used: Western Blot, Mutagenesis, Staining

    20) Product Images from "Cargo binding activates myosin VIIA motor function in cells"

    Article Title: Cargo binding activates myosin VIIA motor function in cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1009188108

    ) Western blotting of each fraction using the antibodies against the marker proteins, Erk (extracellular signal-regulated kinase) (cytoplasmic marker), and N -cadherin (plasma membrane marker). ( B ) MyRip promotes the recruitment of myosin VIIA to membrane vesicles. The amount of myc-M7SAHcoilTail in cytoplasmic fraction (Cy) and small vesicle-containing fraction (V) were analyzed by Western blotting using anti-myc antibodies. Actin staining was done by using anti-actin antibodies as loading control. ( C ) The statistical representation of the effect of MyRip on membrane-vesicle recruitment of myc-M7SAHcoilTail. Band density of small vesicle-containing fraction (V) is denominated with the band density of cytoplasmic fraction (Cy). The band density was quantitated with ImageJ software. The value was normalized by using the transfection efficiency of myc-M7SAHcoilTail and mCherry-MyRip. The value of the cell extracts obtained from cells expressing myc-M7SAHcoilTail alone was taken to be 1. Error bars show ± SD from four independent experiments. ( D ) The effect of MyRip on myosin VIIA dimer formation revealed by chemical cross-linking. ARPE-19 cells were cotransfected with the indicated combinations of plasmids encoding myosin VIIA tail and MyRip. The cell extracts were subjected to cross-linking with 0 or 15 mM EDC/sulfo-NHS ( N -hydroxysulfosuccinimide) (1:1) for 5 min. The products were analyzed by Western blotting using anti-myc antibodies. Dimer and monomer of myc-M7 tail constructs are indicated by arrowheads. The exposure time of monomer bands are shorter than dimer bands. ( E ) The statistical representation of the effect of MyRip on dimer formation. Band density of dimer was denominated with the band density of monomer. The value of the cell extracts obtained from myc-M7SAHcoilTail alone expressing cells was taken to be 1. Error bars show ± SD from three independent experiments. The mean values were normalized by using the cotransfection efficiency of myc-M7SAHcoilTail and mCherry-MyRip.
    Figure Legend Snippet: ) Western blotting of each fraction using the antibodies against the marker proteins, Erk (extracellular signal-regulated kinase) (cytoplasmic marker), and N -cadherin (plasma membrane marker). ( B ) MyRip promotes the recruitment of myosin VIIA to membrane vesicles. The amount of myc-M7SAHcoilTail in cytoplasmic fraction (Cy) and small vesicle-containing fraction (V) were analyzed by Western blotting using anti-myc antibodies. Actin staining was done by using anti-actin antibodies as loading control. ( C ) The statistical representation of the effect of MyRip on membrane-vesicle recruitment of myc-M7SAHcoilTail. Band density of small vesicle-containing fraction (V) is denominated with the band density of cytoplasmic fraction (Cy). The band density was quantitated with ImageJ software. The value was normalized by using the transfection efficiency of myc-M7SAHcoilTail and mCherry-MyRip. The value of the cell extracts obtained from cells expressing myc-M7SAHcoilTail alone was taken to be 1. Error bars show ± SD from four independent experiments. ( D ) The effect of MyRip on myosin VIIA dimer formation revealed by chemical cross-linking. ARPE-19 cells were cotransfected with the indicated combinations of plasmids encoding myosin VIIA tail and MyRip. The cell extracts were subjected to cross-linking with 0 or 15 mM EDC/sulfo-NHS ( N -hydroxysulfosuccinimide) (1:1) for 5 min. The products were analyzed by Western blotting using anti-myc antibodies. Dimer and monomer of myc-M7 tail constructs are indicated by arrowheads. The exposure time of monomer bands are shorter than dimer bands. ( E ) The statistical representation of the effect of MyRip on dimer formation. Band density of dimer was denominated with the band density of monomer. The value of the cell extracts obtained from myc-M7SAHcoilTail alone expressing cells was taken to be 1. Error bars show ± SD from three independent experiments. The mean values were normalized by using the cotransfection efficiency of myc-M7SAHcoilTail and mCherry-MyRip.

    Techniques Used: Western Blot, Marker, Staining, Software, Transfection, Expressing, Construct, Cotransfection

    21) Product Images from "Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1"

    Article Title: Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.5b00903

    Venn diagrams of reactive lysine sites and amine-reactive cross-linking products. (A) Overlap of reactive lysine sites in HOP2− MND1 for EDC, DSS, and BS 2 G (combined BS 2 G d0 and BS 2 G d0d6 ). (B) Identified unique amino acid sites observed for mono-, loop-, and cross-links in HOP2 and MND1.
    Figure Legend Snippet: Venn diagrams of reactive lysine sites and amine-reactive cross-linking products. (A) Overlap of reactive lysine sites in HOP2− MND1 for EDC, DSS, and BS 2 G (combined BS 2 G d0 and BS 2 G d0d6 ). (B) Identified unique amino acid sites observed for mono-, loop-, and cross-links in HOP2 and MND1.

    Techniques Used:

    of the amine-reactive- (A) and EDC- (B) derived cross-links within the three domains of HOP2 (red) and MND1 (brown). Line thickness corresponds to the number of identified cross-link spectra. Lysine positions are marked in yellow. (A) Amine-reactive links: Gray lines correspond to cross-links derived from DSS, whereas green lines indicate BS 2 G cross-linking products. (B) EDC links: Black lines correspond to cross-links derived from EDC. MND1_N: N-terminus of MND1 protein. MND1_CC: coiled-coil region of MND1. MND1_C: C-terminal domain of MND1. HOP2_N: N-terminal domain of HOP2. HOP2_CC: coiled-coil region of HOP2. HOP2_C: C-terminus of HOP2.
    Figure Legend Snippet: of the amine-reactive- (A) and EDC- (B) derived cross-links within the three domains of HOP2 (red) and MND1 (brown). Line thickness corresponds to the number of identified cross-link spectra. Lysine positions are marked in yellow. (A) Amine-reactive links: Gray lines correspond to cross-links derived from DSS, whereas green lines indicate BS 2 G cross-linking products. (B) EDC links: Black lines correspond to cross-links derived from EDC. MND1_N: N-terminus of MND1 protein. MND1_CC: coiled-coil region of MND1. MND1_C: C-terminal domain of MND1. HOP2_N: N-terminal domain of HOP2. HOP2_CC: coiled-coil region of HOP2. HOP2_C: C-terminus of HOP2.

    Techniques Used: Derivative Assay

    22) Product Images from "Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **"

    Article Title: Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **

    Journal: Advanced functional materials

    doi: 10.1002/adfm.201203319

    Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.
    Figure Legend Snippet: Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.

    Techniques Used: Imaging, Fluorescence

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    Modification:

    Article Title: CheZ Has No Effect on Flagellar Motors Activated by CheY13DK106YW
    Article Snippet: .. Protein cross-linking experiments were done with EDC (1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride), NHS ( N -hydroxysuccinimide) (ICN, Aurora, Ohio), or dimethylsuberimidate dihydrochloride (DMS) (Pierce, Rockford, Ill.), according to the procedures of Blat and Eisenbach ( ) and Bren et al. ( ) with the following modification: CheZ cross-linked products were assayed on immunoblots as described above. ..

    Concentration Assay:

    Article Title: Bin1 directly remodels actin dynamics through its BAR domain
    Article Snippet: .. Briefly, F‐actin and hBin1 (isoform 1) or dBAR were mixed at the indicated concentrations in 10 mM PIPES, 50 mM KCl, 2 mM MgCl2 , pH 6.8 and incubated for 30 min, before adding the EDC crosslinker (1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride; Thermo Fisher Scientific) at a final concentration of 1 mM. .. After 30 min of crosslinking, the reaction was stopped by adding SDS‐loading buffer [60 mM Trizma base (pH 6.8), 2% (w/v) SDS, 5% (v/v) mercaptoethanol, 0.01% (w/v) Brilliant blue], and samples were analyzed via SDS–PAGE and Coomassie Blue staining.

    Incubation:

    Article Title: Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD
    Article Snippet: .. After purification, mitochondrial samples were resuspended and incubated with different cross-linking reagents − 1% Formaldehyde in pre-chilled HBS, 10 mM EDC (#22980, Thermo Fisher) in HBS buffer. .. The samples were incubated under 25°C and stopped at different time point by adding SDS-sample buffer and boiling.

    Article Title: A Missense Mutation in a Highly Conserved Alternate Exon of Dynamin-1 Causes Epilepsy in Fitful Mice
    Article Snippet: .. 50 or 100µg of protein was incubated with 0 or 20mM EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride; Pierce) in a final constant volume between samples for 45 minutes at RT in the dark. .. Samples were then diluted in Laemmli buffer, incubated at 95°C for 5 minutes, resolved by SDS-PAGE, transferred to nitrocellulose membrane and processed as for Western blots (see above).

    Article Title: Bin1 directly remodels actin dynamics through its BAR domain
    Article Snippet: .. Briefly, F‐actin and hBin1 (isoform 1) or dBAR were mixed at the indicated concentrations in 10 mM PIPES, 50 mM KCl, 2 mM MgCl2 , pH 6.8 and incubated for 30 min, before adding the EDC crosslinker (1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride; Thermo Fisher Scientific) at a final concentration of 1 mM. .. After 30 min of crosslinking, the reaction was stopped by adding SDS‐loading buffer [60 mM Trizma base (pH 6.8), 2% (w/v) SDS, 5% (v/v) mercaptoethanol, 0.01% (w/v) Brilliant blue], and samples were analyzed via SDS–PAGE and Coomassie Blue staining.

    Purification:

    Article Title: Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD
    Article Snippet: .. After purification, mitochondrial samples were resuspended and incubated with different cross-linking reagents − 1% Formaldehyde in pre-chilled HBS, 10 mM EDC (#22980, Thermo Fisher) in HBS buffer. .. The samples were incubated under 25°C and stopped at different time point by adding SDS-sample buffer and boiling.

    Western Blot:

    Article Title: CheZ Has No Effect on Flagellar Motors Activated by CheY13DK106YW
    Article Snippet: .. Protein cross-linking experiments were done with EDC (1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride), NHS ( N -hydroxysuccinimide) (ICN, Aurora, Ohio), or dimethylsuberimidate dihydrochloride (DMS) (Pierce, Rockford, Ill.), according to the procedures of Blat and Eisenbach ( ) and Bren et al. ( ) with the following modification: CheZ cross-linked products were assayed on immunoblots as described above. ..

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    Thermo Fisher edc
    The content of <t>EdU,</t> <t>EdC</t> and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.
    Edc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edc/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    edc - by Bioz Stars, 2020-09
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    93
    Thermo Fisher 6 cl hobt edc
    Reaction mechanism ( A ) and scheme ( B ) for the linkage-specific sialic acid derivatization using <t>EDC,</t> ethanol and the array of catalysts (in A exemplified by the catalyst <t>HOBt</t> ( Figure 1 )). The expected reaction products included ethyl ester formation on α2,6-linked sialic acids, thereby gaining +28.032 Da ( A and B , top ), while α2,3-linked sialic acids formed lactones under the same conditions (−18.011 Da; B, bottom ). Misconversion leads to ethyl esterification on α2,3-linked sialic acids or lactone formation on α2,6-linked sialic acids with the same changes in mass as indicated above.
    6 Cl Hobt Edc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 cl hobt edc/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    6 cl hobt edc - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    The content of EdU, EdC and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.

    Journal: Open Biology

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    doi: 10.1098/rsob.150172

    Figure Lengend Snippet: The content of EdU, EdC and their metabolites in HeLa cells incubated with EdU or EdC. ( a ) The amount of particular nucleosides and nucleotides in HeLa and 143B cells incubated with 10 µM EdU or EdC for 4 h. ND, non-detected. The data are presented as mean ± s.e.m. ( b ) The amount of thymidine and its nucleotides in HeLa and 143B cells. The data are presented as mean ± s.e.m.

    Article Snippet: EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex).

    Techniques: Incubation

    The impact of THU and specific siRNAs on the incorporation of EdU into DNA. ( a ) The EdU-derived signal intensity in HeLa cells incubated for 2 h with EdC or EdU in the presence of THU. For the detection of EdU, a click reaction was used. The data are normalized to percentage of the signal of control cells incubated with EdC or EdU without THU (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( b ) The impact of siRNA against CDD and DCTD on the incorporation of EdU into the DNA in cells incubated for 2 h with EdU or EdC. For the detection of EdU, a click reaction was used. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( c ) The amount of CDD and DCTD measured by immunoblots in cells treated with siRNA against CDD and DCTD. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m.

    Journal: Open Biology

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    doi: 10.1098/rsob.150172

    Figure Lengend Snippet: The impact of THU and specific siRNAs on the incorporation of EdU into DNA. ( a ) The EdU-derived signal intensity in HeLa cells incubated for 2 h with EdC or EdU in the presence of THU. For the detection of EdU, a click reaction was used. The data are normalized to percentage of the signal of control cells incubated with EdC or EdU without THU (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( b ) The impact of siRNA against CDD and DCTD on the incorporation of EdU into the DNA in cells incubated for 2 h with EdU or EdC. For the detection of EdU, a click reaction was used. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m. ( c ) The amount of CDD and DCTD measured by immunoblots in cells treated with siRNA against CDD and DCTD. The data were normalized to percentage of the signal of cells incubated with control siRNA (equal to 100%, not shown). The data are presented as mean ± s.e.m.

    Article Snippet: EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex).

    Techniques: Derivative Assay, Incubation, Western Blot

    The microscopy analysis of EdC conversion to EdU using an antibody reaction. ( a ) Fluorescence detection of EdU by means of an anti-bromodeoxyuridine antibody (clone B44). HeLa cells were incubated with either 10 µM EdU or 10 µM EdC. Then, the detection of EdU (in green) and DNA using DAPI (in blue) was performed. ( b ) The analysis of the reactivity of anti-bromodeoxyuridine antibody (clone B44) using EdU with biotin at the 5′ end and EdC with biotin at the 3′ or 5′ end. The data were normalized to percentage of the signal provided by EdU (equal to 100%). The data are presented as mean ± s.e.m.

    Journal: Open Biology

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    doi: 10.1098/rsob.150172

    Figure Lengend Snippet: The microscopy analysis of EdC conversion to EdU using an antibody reaction. ( a ) Fluorescence detection of EdU by means of an anti-bromodeoxyuridine antibody (clone B44). HeLa cells were incubated with either 10 µM EdU or 10 µM EdC. Then, the detection of EdU (in green) and DNA using DAPI (in blue) was performed. ( b ) The analysis of the reactivity of anti-bromodeoxyuridine antibody (clone B44) using EdU with biotin at the 5′ end and EdC with biotin at the 3′ or 5′ end. The data were normalized to percentage of the signal provided by EdU (equal to 100%). The data are presented as mean ± s.e.m.

    Article Snippet: EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex).

    Techniques: Microscopy, Fluorescence, Incubation

    Run-on replication assay and hypotonic introduction of EdUTP and EdCTP, EdCDP and EdCMP. ( a ) The detection of EdU and EdC using Alexa Fluor 488 azide in permeabilized HeLa cells (in green). The nuclear DNA was stained by DAPI (in blue). ( b ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdUTP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdUTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( c ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdCMP, EdCDP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdCMP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( d ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide in HeLa cells after the hypotonic introduction of EdUTP or EdCTP with the concurrent introduction of dTTP. The data are normalized to percentage of the signal of EdUTP- or EdCTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m.

    Journal: Open Biology

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    doi: 10.1098/rsob.150172

    Figure Lengend Snippet: Run-on replication assay and hypotonic introduction of EdUTP and EdCTP, EdCDP and EdCMP. ( a ) The detection of EdU and EdC using Alexa Fluor 488 azide in permeabilized HeLa cells (in green). The nuclear DNA was stained by DAPI (in blue). ( b ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdUTP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdUTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( c ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide or EdU by the anti-bromodeoxyuridine antibody clone B44 in HeLa cells after the hypotonic introduction of EdCMP, EdCDP and EdCTP followed by a 30-min incubation in medium. The data are normalized to percentage of the signal of EdCMP-treated cells (equal to 100%). The data are presented as mean ± s.e.m. ( d ) The average signal in cell nuclei after detection of EdU and EdC by Alexa Fluor 488 azide in HeLa cells after the hypotonic introduction of EdUTP or EdCTP with the concurrent introduction of dTTP. The data are normalized to percentage of the signal of EdUTP- or EdCTP-treated cells (equal to 100%). The data are presented as mean ± s.e.m.

    Article Snippet: EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex).

    Techniques: Staining, Incubation

    EdU- and dT-content ratios and the dependence of EdU incorporation on EdC concentrations. ( a ) The ratio between the content of EdU and dT in isolated DNA after a 24-h incubation with 10 µM EdU or EdC in five cell lines is shown. ( b ) The average nuclear signal in five cell lines incubated with 0.016–250 µM EdC for 4 h. The detection of the signal was performed using a click reaction. The data were normalized to the signal provided by 250 µM EdC (equal to 100%). The data are presented as mean ± s.e.m.

    Journal: Open Biology

    Article Title: Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    doi: 10.1098/rsob.150172

    Figure Lengend Snippet: EdU- and dT-content ratios and the dependence of EdU incorporation on EdC concentrations. ( a ) The ratio between the content of EdU and dT in isolated DNA after a 24-h incubation with 10 µM EdU or EdC in five cell lines is shown. ( b ) The average nuclear signal in five cell lines incubated with 0.016–250 µM EdC for 4 h. The detection of the signal was performed using a click reaction. The data were normalized to the signal provided by 250 µM EdC (equal to 100%). The data are presented as mean ± s.e.m.

    Article Snippet: EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex).

    Techniques: Isolation, Incubation

    QCM monitoring and SPR sensorgram evidencing all steps involved for the detection of the interaction between sUA and Naip1 protein. (A) QCM responses over time within sUA injection after Naip1 immobilization upon anti-GFP adsorption on the gold quartz crystals surface at 37°C. The arrows indicate sample injection. (B) Schematic representation of the constructed SPR sensor chip (in the box). Sequential addition of compounds into the system: (i) addition of the buffer solution (PBS, 10 mmol L −1 at pH 7.4); (ii) mixture consisting of EDC (150 mmol L −1 ) and NHS (150 mmol L −1 ); (iii) PBS; (iv) immobilization of anti-GFP (10 μg mL −1 ); (v) PBS; (vi) addition of ethanolamine (EA); addition of cell lysates containing Naip1 protein (2 μg mol L −1 ). It is possible observe a very intensive response for the interaction of the Naip1 protein with anti-GFP; (v) PBS;(vi) addition of pure H 2 O; (vii) addition of sUA (2 μmol L −1 , purple line) and palmitate (2 μmol L −1 , green line). It is possible to observe the significant variation of the SPR angle (Δθ SPR ) due to the interaction between sUA and Naip protein. In A and B, data are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: Sensing soluble uric acid by Naip1-Nlrp3 platform

    doi: 10.1101/2020.05.15.077644

    Figure Lengend Snippet: QCM monitoring and SPR sensorgram evidencing all steps involved for the detection of the interaction between sUA and Naip1 protein. (A) QCM responses over time within sUA injection after Naip1 immobilization upon anti-GFP adsorption on the gold quartz crystals surface at 37°C. The arrows indicate sample injection. (B) Schematic representation of the constructed SPR sensor chip (in the box). Sequential addition of compounds into the system: (i) addition of the buffer solution (PBS, 10 mmol L −1 at pH 7.4); (ii) mixture consisting of EDC (150 mmol L −1 ) and NHS (150 mmol L −1 ); (iii) PBS; (iv) immobilization of anti-GFP (10 μg mL −1 ); (v) PBS; (vi) addition of ethanolamine (EA); addition of cell lysates containing Naip1 protein (2 μg mol L −1 ). It is possible observe a very intensive response for the interaction of the Naip1 protein with anti-GFP; (v) PBS;(vi) addition of pure H 2 O; (vii) addition of sUA (2 μmol L −1 , purple line) and palmitate (2 μmol L −1 , green line). It is possible to observe the significant variation of the SPR angle (Δθ SPR ) due to the interaction between sUA and Naip protein. In A and B, data are representative of three independent experiments.

    Article Snippet: Afterward, gold crystals were incubated with EDC (100 mmol/L) and NHS (150 mmol/L) and then, 200 μL of anti-GFP (MA5-15256, Thermo Fisher Scientific), at 20 μg/mL, diluted in ultrapure water was deposited over each crystal for 16 h at 4 °C in a humid chamber.

    Techniques: SPR Assay, Injection, Adsorption, Construct, Chromatin Immunoprecipitation

    Schematic Illustration of “Post-Fabrication” siRNA Loading and Surface Modification: (a) NHS-PEG 3.4k -COOH, DMF, Pyridine; (b) SPDP, PBS/CH 3 CN; (c) siRNA-SH, PBS; (d) Poly-L-lysine, EDC, Sulfo-NHS, PBS

    Journal: Molecular pharmaceutics

    Article Title: Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo

    doi: 10.1021/acs.molpharmaceut.5b00054

    Figure Lengend Snippet: Schematic Illustration of “Post-Fabrication” siRNA Loading and Surface Modification: (a) NHS-PEG 3.4k -COOH, DMF, Pyridine; (b) SPDP, PBS/CH 3 CN; (c) siRNA-SH, PBS; (d) Poly-L-lysine, EDC, Sulfo-NHS, PBS

    Article Snippet: EDC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride), hydrox-ylamine hydrochloride and SPDP were from Thermo Scientific.

    Techniques: Modification

    Reaction mechanism ( A ) and scheme ( B ) for the linkage-specific sialic acid derivatization using EDC, ethanol and the array of catalysts (in A exemplified by the catalyst HOBt ( Figure 1 )). The expected reaction products included ethyl ester formation on α2,6-linked sialic acids, thereby gaining +28.032 Da ( A and B , top ), while α2,3-linked sialic acids formed lactones under the same conditions (−18.011 Da; B, bottom ). Misconversion leads to ethyl esterification on α2,3-linked sialic acids or lactone formation on α2,6-linked sialic acids with the same changes in mass as indicated above.

    Journal: Molecules

    Article Title: Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization

    doi: 10.3390/molecules24193617

    Figure Lengend Snippet: Reaction mechanism ( A ) and scheme ( B ) for the linkage-specific sialic acid derivatization using EDC, ethanol and the array of catalysts (in A exemplified by the catalyst HOBt ( Figure 1 )). The expected reaction products included ethyl ester formation on α2,6-linked sialic acids, thereby gaining +28.032 Da ( A and B , top ), while α2,3-linked sialic acids formed lactones under the same conditions (−18.011 Da; B, bottom ). Misconversion leads to ethyl esterification on α2,3-linked sialic acids or lactone formation on α2,6-linked sialic acids with the same changes in mass as indicated above.

    Article Snippet: Briefly, 20 µL of HOBt + EDC, 6-Cl-HOBt + EDC, + 6-CF3 -HOBt + EDC, or HOAt + EDC in ethanol was added to the wells of a 96-well NUNC V-bottom plate (Thermo Scientific, Waltham, MA, USA).

    Techniques:

    Representative MALDI-TOF-MS spectrum of derivatized N -glycans released from total plasma (TPNG) using 6-Cl-HOBt as catalyst at native conditions, and linkage-specificity of the reaction conditions as determined on a TPNG sample. ( A ) Mass spectrum showing relative intensities from m / z 1000 to 3700. Compositional assignments were based on accurate mass and isotopic pattern matching, structural assignments were based on literature and biosynthetic pathways. Except for the N -acetylneuraminic acids, glycosidic linkages were not determined. The assigned glycan signals had a signal-to-noise (S/N) > 9. Different sialic acid linkages are indicated by a left (α2,3) or right (α2,6) angle. ( B ) Relative ratio of di- and triantennary sialylated species with the gross composition hexose (H)5; N -acetylhexosamine (N)4; N -acetylneuraminic acids (S)2; and H6N5S3 and their different sialic acid linkage variants (lactonized α2,3-sialic acid: L; ethyl esterified α2,6-sialic acid: E) after incubation with the different catalysts under native and acidic pH at 37 °C. Note: the di- and triantennary glycans are total area normalized to 100% separately. Error bars represent SD for triplicate measurements.

    Journal: Molecules

    Article Title: Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization

    doi: 10.3390/molecules24193617

    Figure Lengend Snippet: Representative MALDI-TOF-MS spectrum of derivatized N -glycans released from total plasma (TPNG) using 6-Cl-HOBt as catalyst at native conditions, and linkage-specificity of the reaction conditions as determined on a TPNG sample. ( A ) Mass spectrum showing relative intensities from m / z 1000 to 3700. Compositional assignments were based on accurate mass and isotopic pattern matching, structural assignments were based on literature and biosynthetic pathways. Except for the N -acetylneuraminic acids, glycosidic linkages were not determined. The assigned glycan signals had a signal-to-noise (S/N) > 9. Different sialic acid linkages are indicated by a left (α2,3) or right (α2,6) angle. ( B ) Relative ratio of di- and triantennary sialylated species with the gross composition hexose (H)5; N -acetylhexosamine (N)4; N -acetylneuraminic acids (S)2; and H6N5S3 and their different sialic acid linkage variants (lactonized α2,3-sialic acid: L; ethyl esterified α2,6-sialic acid: E) after incubation with the different catalysts under native and acidic pH at 37 °C. Note: the di- and triantennary glycans are total area normalized to 100% separately. Error bars represent SD for triplicate measurements.

    Article Snippet: Briefly, 20 µL of HOBt + EDC, 6-Cl-HOBt + EDC, + 6-CF3 -HOBt + EDC, or HOAt + EDC in ethanol was added to the wells of a 96-well NUNC V-bottom plate (Thermo Scientific, Waltham, MA, USA).

    Techniques: Mass Spectrometry, Incubation

    Chemical structure of the catalysts evaluated in this study. HOBt: 1-hydroxybenzotriazole, 6-Cl-HOBt: 6-chloro-1-hydroxybenzotriazole, 6-CF 3 -HOBt: 1-hydroxy-6-(trifluoromethyl)benzotriazole, HOAt: 3-hydroxytriazolo[4,5-b]pyridine, HOCt: ethyl 1-hydroxytriazole-4-carboxylate, Oxyma Pure: ethyl (2E)-2-cyano-2-hydroxyiminoacetate.

    Journal: Molecules

    Article Title: Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization

    doi: 10.3390/molecules24193617

    Figure Lengend Snippet: Chemical structure of the catalysts evaluated in this study. HOBt: 1-hydroxybenzotriazole, 6-Cl-HOBt: 6-chloro-1-hydroxybenzotriazole, 6-CF 3 -HOBt: 1-hydroxy-6-(trifluoromethyl)benzotriazole, HOAt: 3-hydroxytriazolo[4,5-b]pyridine, HOCt: ethyl 1-hydroxytriazole-4-carboxylate, Oxyma Pure: ethyl (2E)-2-cyano-2-hydroxyiminoacetate.

    Article Snippet: Briefly, 20 µL of HOBt + EDC, 6-Cl-HOBt + EDC, + 6-CF3 -HOBt + EDC, or HOAt + EDC in ethanol was added to the wells of a 96-well NUNC V-bottom plate (Thermo Scientific, Waltham, MA, USA).

    Techniques:

    Representative MALDI-TOF-MS spectra of derivatized SL standards under different conditions using HOBt as catalyst. 2,3-SL ( A – C ) and 2,6-SL ( D – F ) incubated for 1 h at 37 °C in ethanol, with EDC and HOBt, at native ( B , E ) low ( A , D ) or high ( C , F ) pH. Lactonized reaction product: [M + Na] + = 638.190 Da, ethyl esterified reaction product: [M + Na] + = 684.232 Da. *: reaction product derived from misconversion of the standard (i.e., ethyl esterification of 2,3-SL or lactonization of 2,6-SL), standard impurity (reported purity: ≥98%), or a combination thereof. Symbols indicate the monosaccharide residues glucose (blue circle), galactose (yellow circle), and N -acetylneuraminic acid (purple diamond). In case of derivatized sialic acids, an α2,3-linkage is indicated by a left angle, an α2,6-linkage by a right angle, while non-derivatized sialic acids are depicted without an angle. Note: mass spectra are main peak normalized.

    Journal: Molecules

    Article Title: Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization

    doi: 10.3390/molecules24193617

    Figure Lengend Snippet: Representative MALDI-TOF-MS spectra of derivatized SL standards under different conditions using HOBt as catalyst. 2,3-SL ( A – C ) and 2,6-SL ( D – F ) incubated for 1 h at 37 °C in ethanol, with EDC and HOBt, at native ( B , E ) low ( A , D ) or high ( C , F ) pH. Lactonized reaction product: [M + Na] + = 638.190 Da, ethyl esterified reaction product: [M + Na] + = 684.232 Da. *: reaction product derived from misconversion of the standard (i.e., ethyl esterification of 2,3-SL or lactonization of 2,6-SL), standard impurity (reported purity: ≥98%), or a combination thereof. Symbols indicate the monosaccharide residues glucose (blue circle), galactose (yellow circle), and N -acetylneuraminic acid (purple diamond). In case of derivatized sialic acids, an α2,3-linkage is indicated by a left angle, an α2,6-linkage by a right angle, while non-derivatized sialic acids are depicted without an angle. Note: mass spectra are main peak normalized.

    Article Snippet: Briefly, 20 µL of HOBt + EDC, 6-Cl-HOBt + EDC, + 6-CF3 -HOBt + EDC, or HOAt + EDC in ethanol was added to the wells of a 96-well NUNC V-bottom plate (Thermo Scientific, Waltham, MA, USA).

    Techniques: Mass Spectrometry, Incubation, Derivative Assay