edc  (Thermo Fisher)


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    Name:
    Pierce EDC No Weigh Format
    Description:
    Thermo Scientific Pierce EDC is a carboxyl and amine reactive zero length crosslinker EDC reacts with a carboxyl group first and forms an amine reactive O acylisourea intermediate that quickly reacts with an amino group to form an amide bond with release of an isourea by product The intermediate is unstable in aqueous solutions and so two step conjugation procedures rely on N hydroxysuccinimide NHS for stabilization Failure to react with an amine will result in hydrolysis of the intermediate regeneration of the carboxyl and release of an N substituted urea A side reaction is the formation of an N acylurea which is usually restricted to carboxyls located in hydrophobic regions of proteins Thermo Scientific No Weigh products are specialty reagents provided in a pre aliquoted format The pre weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial The format enables use of a fresh vial of reagent each time eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability 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
    Catalog Number:
    a35391
    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 carboxyl and amine reactive zero length crosslinker EDC reacts with a carboxyl group first and forms an amine reactive O acylisourea intermediate that quickly reacts with an amino group to form an amide bond with release of an isourea by product The intermediate is unstable in aqueous solutions and so two step conjugation procedures rely on N hydroxysuccinimide NHS for stabilization Failure to react with an amine will result in hydrolysis of the intermediate regeneration of the carboxyl and release of an N substituted urea A side reaction is the formation of an N acylurea which is usually restricted to carboxyls located in hydrophobic regions of proteins Thermo Scientific No Weigh products are specialty reagents provided in a pre aliquoted format The pre weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial The format enables use of a fresh vial of reagent each time eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability 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
    https://www.bioz.com/result/edc/product/Thermo Fisher
    Average 99 stars, based on 44 article reviews
    Price from $9.99 to $1999.99
    edc - by Bioz Stars, 2020-01
    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 "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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    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 "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

    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 "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

    12) 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

    Related Articles

    Centrifugation:

    Article Title: Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
    Article Snippet: After resuspension, 3.3 μL of 300 μmol/L 5′-amine-modified EraCode DNA oligonucleotide and 5.0 μL of freshly prepared 10 g/L EDC (Pierce Chemical) were added. .. The reaction was mixed thoroughly and incubated at room temperature in the dark for 30 min. A second 5.0 μL of 10 g/L EDC was added, and the mixture was incubated for 30 min. EraCode microspheres were washed by the addition of 1.0 mL of 10 mmol/L Tris (pH 8.0) containing 0.2 mL/L Tween 20 and pelleted by centrifugation at 8000 g for 1 min; the supernatant was then removed.

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer. .. Cross-linking was quenched with the addition of β-mercaptoethanol (final concentration 20 mM, Sigma) and 4× sample buffer.

    Expressing:

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: Cross-linking of EGFP-EVH2 (Mena) in lysates MVD7 cells stably expressing EGFP-EVH2 (Mena) were lysed in a modified IP lysis buffer [10% glycerol, 1% IGEPAL CA-630, 15 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM MES (pH 6.0), 40 mM β-glycerophosphate, 500mM sodium chloride, 2mM magnesium chloride and protease inhibitor tablet without EDTA (Roche)]. .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer.

    Stable Transfection:

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: Cross-linking of EGFP-EVH2 (Mena) in lysates MVD7 cells stably expressing EGFP-EVH2 (Mena) were lysed in a modified IP lysis buffer [10% glycerol, 1% IGEPAL CA-630, 15 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM MES (pH 6.0), 40 mM β-glycerophosphate, 500mM sodium chloride, 2mM magnesium chloride and protease inhibitor tablet without EDTA (Roche)]. .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer.

    Synthesized:

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. Purification of the synthesized ILO-PC was performed on an SPE cartridge (SupelClean LC18, Sigma-Aldrich, St. Louis, MO) using a gradient of methanol in water.

    Autoradiography:

    Article Title: Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5 *
    Article Snippet: EDC was obtained from Pierce, and SartoriusTM Vivapure-S spin columns and plasmid pGro7 were purchased from Thermo Fisher Scientific (Waltham, MA). .. PVDF membrane was obtained from Millipore (Billerica, MA); Clarity Western ECL reagent was obtained from Bio-Rad, and Blue Devil autoradiography film was acquired from Genesee Scientific (San Diego).

    Adsorption:

    Article Title: CD44-mediated Adhesion to Hyaluronic Acid Contributes to Mechanosensing and Invasive Motility
    Article Snippet: .. Since the hydrogels are resistant to passive protein adsorption, crosslinked hydrogels were conjugated with poly-L-lysine by carbodiimide chemistry using 0.5 M EDC (Pierce) and 0.5 M NHS (Sigma) in 0.1 M MES buffer at pH 5.8. .. After rinsing, a solution of 0.5% poly-L-lysine (Sigma) in PBS was added for 1.5 h, then a 0.1 mg/mL solution of human plasma fibronectin (Millipore) or rat laminin (Life Technologies) was adsorbed for 1 h at room temperature.

    Incubation:

    Article Title: Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
    Article Snippet: After resuspension, 3.3 μL of 300 μmol/L 5′-amine-modified EraCode DNA oligonucleotide and 5.0 μL of freshly prepared 10 g/L EDC (Pierce Chemical) were added. .. The reaction was mixed thoroughly and incubated at room temperature in the dark for 30 min. A second 5.0 μL of 10 g/L EDC was added, and the mixture was incubated for 30 min. EraCode microspheres were washed by the addition of 1.0 mL of 10 mmol/L Tris (pH 8.0) containing 0.2 mL/L Tween 20 and pelleted by centrifugation at 8000 g for 1 min; the supernatant was then removed.

    Article Title: A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity
    Article Snippet: .. 10 μL 50 mg/ml Sulfo-NHS (Pierce) and 10 μL 50 mg/ml EDC (Pierce) were added and incubated at 25 °C for 20 min while shaking. ..

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer. .. Cross-linking was quenched with the addition of β-mercaptoethanol (final concentration 20 mM, Sigma) and 4× sample buffer.

    Article Title: Nanoliter high throughput quantitative PCR
    Article Snippet: The sheet is next slowly passed through a layer of 30 ml of an oxidation solution (5 mM KMnO4 and 19.5 mM NaIO4 ) floating on 1l of Fluorinert™ (FC3283), incubated for 2 h, rinsed in RODI water and dried in a stream of dry nitrogen gas. .. After oxidation, a hydrophilic PEG layer is deposited inside the through-holes by repeating the previous steps except replacing the oxidation solution with 30 ml of 15 mg/ml EDC (Pierce) and 5 mg/ml PEG 5000 (Nektar-Synasia) in HEPES buffer (pH 7.5).

    Article Title: A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein σ1
    Article Snippet: A one-step cross-linking procedure using EDC (Pierce) was employed to couple σ1 to the AFM tips ( ) ( ). .. Tips were dried, treated with a 4% solution of 3-aminopropyltriethoxysilane (APTES, Sigma) in acetone for 3 min, rinsed three times in acetone, and incubated in a 2 mg/ml solution of BS3 (Pierce) for 30 min.

    Article Title: Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex *
    Article Snippet: The natively eluted Nup84 complex (200 μl) was cross-linked via the addition of isotopically labeled DSS (d0:d12 = 1:1, Creative Moleculesan an online company located at: ) to yield a final concentration of 1 m m and incubated for 45 min at 25 °C and 750 rpm of agitation. .. In the case of cross-linking using EDC (Pierce), the sample was equilibrated and eluted in EDC cross-linking buffer (20 m m MES, pH 6.5, 500 m m NaCl, 2 m m MgCl2 , 0.1% CHAPS, 1 m m DTT); EDC was added to the sample to yield a final concentration of 25 m m , and N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) was added to yield a final concentration of 0.5 m m ( i.e. 2% molar ratio with respect to EDC).

    Article Title: RanGTP Targets p97 to RanBP2, a Filamentous Protein Localized at the Cytoplasmic Periphery of the Nuclear Pore Complex
    Article Snippet: After coating, the plates were incubated overnight with binding buffer (3% BSA and 0.1% Tween 20 in coating buffer). .. For analysis of p97 binding, proteins were cross-linked for 15 min with 1 mg/ml EDC ( Pierce Chemical , Rockford, IL) in the same buffer.

    Article Title: Depletion of Cholinergic Amacrine Cells by a Novel Immunotoxin Does Not Perturb the Formation of Segregated On and Off Cone Bipolar Cell Projections
    Article Snippet: .. Saporin (Sigma, St. Louis, MO) was dissolved in conjugation buffer [0.1 m 2-( N -morpholino) ethanesulfonic acid, 0.9 m NaCl, pH 4.7] and mixed with anti-VAChT antibody (Chemicon International, Temecula, CA) in equal amounts and incubated at room temperature for 2 hr in the presence of EDC (Pierce, Rockford, IL) following the vendor's instructions. ..

    Mass Spectrometry:

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. The purity of the synthesized product was checked by TLC on Kieselgel 60 plates in chloroform-methanol-water (100:50:10, v/v/v) by staining with 10% CuSO4/8.5% H3 PO4 and followed by heating to 120 °C as well as by mass-spectrometry using Sciex 6500 QTRAP triple quadrupole ion trap hybrid mass spectrometer interfaced with Agilent 1290 UHPLC system.

    Modification:

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: Cross-linking of EGFP-EVH2 (Mena) in lysates MVD7 cells stably expressing EGFP-EVH2 (Mena) were lysed in a modified IP lysis buffer [10% glycerol, 1% IGEPAL CA-630, 15 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM MES (pH 6.0), 40 mM β-glycerophosphate, 500mM sodium chloride, 2mM magnesium chloride and protease inhibitor tablet without EDTA (Roche)]. .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer.

    Western Blot:

    Article Title: A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product
    Article Snippet: Western blots were probed with a high affinity rat antibody directed against the HA epitope (clone 3F10; Roche Molecular Biochemicals). .. Chemical cross-linking was performed by treating axoneme samples with EDC (Pierce Chemical Co.) for 1 h at room temperature ( ).

    Article Title: Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5 *
    Article Snippet: EDC was obtained from Pierce, and SartoriusTM Vivapure-S spin columns and plasmid pGro7 were purchased from Thermo Fisher Scientific (Waltham, MA). .. PVDF membrane was obtained from Millipore (Billerica, MA); Clarity Western ECL reagent was obtained from Bio-Rad, and Blue Devil autoradiography film was acquired from Genesee Scientific (San Diego).

    Conjugation Assay:

    Article Title: Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
    Article Snippet: For each conjugation reaction, a mixture (400 μL) containing ~5 × 106 carboxylated microspheres (Luminex Corporation) was centrifuged at 8000 g for 1 min. .. After resuspension, 3.3 μL of 300 μmol/L 5′-amine-modified EraCode DNA oligonucleotide and 5.0 μL of freshly prepared 10 g/L EDC (Pierce Chemical) were added.

    Article Title: Rapid Nanoparticle-Mediated Monitoring of Bacterial Metabolic Activity and Assessment of Antimicrobial Susceptibility in Blood with Magnetic Relaxation
    Article Snippet: Paragraph title: Conjugation of Concanavalin A to aminated silica-coated iron oxide nanoparticles ... Initially, in 1 mL of cold MES buffer (0.1 M, pH 6.0) 4.8 mg EDC (Pierce) and 3 mg NHS (Pierce) were dissolved.

    Article Title: Depletion of Cholinergic Amacrine Cells by a Novel Immunotoxin Does Not Perturb the Formation of Segregated On and Off Cone Bipolar Cell Projections
    Article Snippet: .. Saporin (Sigma, St. Louis, MO) was dissolved in conjugation buffer [0.1 m 2-( N -morpholino) ethanesulfonic acid, 0.9 m NaCl, pH 4.7] and mixed with anti-VAChT antibody (Chemicon International, Temecula, CA) in equal amounts and incubated at room temperature for 2 hr in the presence of EDC (Pierce, Rockford, IL) following the vendor's instructions. ..

    Flow Cytometry:

    Article Title: Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration
    Article Snippet: The collagen completely dried under a laminar flow hood overnight and rinsed several times with MilliQ water afterwards. .. The collagen grafts then received a cross-linking treatment of 50 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, EDC, (Pierce) in 100% ethanol, agitated overnight at room temperature.

    Protease Inhibitor:

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: Cross-linking of EGFP-EVH2 (Mena) in lysates MVD7 cells stably expressing EGFP-EVH2 (Mena) were lysed in a modified IP lysis buffer [10% glycerol, 1% IGEPAL CA-630, 15 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM MES (pH 6.0), 40 mM β-glycerophosphate, 500mM sodium chloride, 2mM magnesium chloride and protease inhibitor tablet without EDTA (Roche)]. .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer.

    Cell Culture:

    Article Title: Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration
    Article Snippet: The collagen grafts then received a cross-linking treatment of 50 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, EDC, (Pierce) in 100% ethanol, agitated overnight at room temperature. .. In preparation for cell culture or mechanical testing, the grafts were rehydrated in PBS.

    other:

    Article Title: Functional ?7-containing nicotinic acetylcholine receptors localize to cell bodies and proximal dendrites in the rat substantia nigra pars reticulata
    Article Snippet: EDC was obtained from Pierce (Rockford, IL, USA).

    Sonication:

    Article Title: Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
    Article Snippet: After resuspension, 3.3 μL of 300 μmol/L 5′-amine-modified EraCode DNA oligonucleotide and 5.0 μL of freshly prepared 10 g/L EDC (Pierce Chemical) were added. .. EraCode microspheres were washed a second time with 1 g/L sodium dodecyl sulfate in 10 mmol/L Tris (pH 8.0) and resuspended in 100 μL of bead buffer (10 mmol/L MOPS, pH 7.5; 1 mmol/L EDTA, 0.5 g/L sodium dodecyl sulfate, 200 mmol/L sodium chloride, and 0.1 g/L sonicated herring sperm DNA).

    Binding Assay:

    Article Title: RanGTP Targets p97 to RanBP2, a Filamentous Protein Localized at the Cytoplasmic Periphery of the Nuclear Pore Complex
    Article Snippet: .. For analysis of p97 binding, proteins were cross-linked for 15 min with 1 mg/ml EDC ( Pierce Chemical , Rockford, IL) in the same buffer. ..

    Labeling:

    Article Title: Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex *
    Article Snippet: The natively eluted Nup84 complex (200 μl) was cross-linked via the addition of isotopically labeled DSS (d0:d12 = 1:1, Creative Moleculesan an online company located at: ) to yield a final concentration of 1 m m and incubated for 45 min at 25 °C and 750 rpm of agitation. .. In the case of cross-linking using EDC (Pierce), the sample was equilibrated and eluted in EDC cross-linking buffer (20 m m MES, pH 6.5, 500 m m NaCl, 2 m m MgCl2 , 0.1% CHAPS, 1 m m DTT); EDC was added to the sample to yield a final concentration of 25 m m , and N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) was added to yield a final concentration of 0.5 m m ( i.e. 2% molar ratio with respect to EDC).

    Purification:

    Article Title: A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity
    Article Snippet: Protein coupling to Luminex beads MagPlex microspheres (Luminex) with different spectral properties were each coupled separately to purified proteins. .. 10 μL 50 mg/ml Sulfo-NHS (Pierce) and 10 μL 50 mg/ml EDC (Pierce) were added and incubated at 25 °C for 20 min while shaking.

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. Purification of the synthesized ILO-PC was performed on an SPE cartridge (SupelClean LC18, Sigma-Aldrich, St. Louis, MO) using a gradient of methanol in water.

    Article Title: Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex *
    Article Snippet: Paragraph title: Purification and Chemical Cross-linking of the Endogenous Nup84 Complex ... In the case of cross-linking using EDC (Pierce), the sample was equilibrated and eluted in EDC cross-linking buffer (20 m m MES, pH 6.5, 500 m m NaCl, 2 m m MgCl2 , 0.1% CHAPS, 1 m m DTT); EDC was added to the sample to yield a final concentration of 25 m m , and N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) was added to yield a final concentration of 0.5 m m ( i.e. 2% molar ratio with respect to EDC).

    Article Title: RanGTP Targets p97 to RanBP2, a Filamentous Protein Localized at the Cytoplasmic Periphery of the Nuclear Pore Complex
    Article Snippet: Microtiter plates were coated with 2.5 ng purified RanBP2 or 25 ng GST-p62 per well by incubation for 24 h in coating buffer (phosphate-buffered saline [PBS] plus 4 mM DTT and 2 μg/ml of the protease inhibitors [E64, phenylmethylsulfonylfluoride, apoprotinin, leupeptin, and pepstatin]). .. For analysis of p97 binding, proteins were cross-linked for 15 min with 1 mg/ml EDC ( Pierce Chemical , Rockford, IL) in the same buffer.

    Staining:

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. The purity of the synthesized product was checked by TLC on Kieselgel 60 plates in chloroform-methanol-water (100:50:10, v/v/v) by staining with 10% CuSO4/8.5% H3 PO4 and followed by heating to 120 °C as well as by mass-spectrometry using Sciex 6500 QTRAP triple quadrupole ion trap hybrid mass spectrometer interfaced with Agilent 1290 UHPLC system.

    Article Title: A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product
    Article Snippet: Gels were stained directly or transferred to Immobilon-P (Millipore). .. Chemical cross-linking was performed by treating axoneme samples with EDC (Pierce Chemical Co.) for 1 h at room temperature ( ).

    SDS Page:

    Article Title: Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex *
    Article Snippet: In the case of cross-linking using EDC (Pierce), the sample was equilibrated and eluted in EDC cross-linking buffer (20 m m MES, pH 6.5, 500 m m NaCl, 2 m m MgCl2 , 0.1% CHAPS, 1 m m DTT); EDC was added to the sample to yield a final concentration of 25 m m , and N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) was added to yield a final concentration of 0.5 m m ( i.e. 2% molar ratio with respect to EDC). .. The cross-linked samples were either directly processed for in-solution digestion or precipitated using 90% cold methanol and resuspended in SDS-PAGE loading buffer for in-gel separation and digestion.

    Plasmid Preparation:

    Article Title: Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5 *
    Article Snippet: .. EDC was obtained from Pierce, and SartoriusTM Vivapure-S spin columns and plasmid pGro7 were purchased from Thermo Fisher Scientific (Waltham, MA). .. PVDF membrane was obtained from Millipore (Billerica, MA); Clarity Western ECL reagent was obtained from Bio-Rad, and Blue Devil autoradiography film was acquired from Genesee Scientific (San Diego).

    Irradiation:

    Article Title: A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein σ1
    Article Snippet: A one-step cross-linking procedure using EDC (Pierce) was employed to couple σ1 to the AFM tips ( ) ( ). .. Soft silicon nitride tips (Veeco, Santa Barbara, CA) were UV irradiated for 15 min and treated with a mixture of 30% H2 O2 /70% H2 SO4 for 30 min.

    Spectrophotometry:

    Article Title: Depletion of Cholinergic Amacrine Cells by a Novel Immunotoxin Does Not Perturb the Formation of Segregated On and Off Cone Bipolar Cell Projections
    Article Snippet: Saporin (Sigma, St. Louis, MO) was dissolved in conjugation buffer [0.1 m 2-( N -morpholino) ethanesulfonic acid, 0.9 m NaCl, pH 4.7] and mixed with anti-VAChT antibody (Chemicon International, Temecula, CA) in equal amounts and incubated at room temperature for 2 hr in the presence of EDC (Pierce, Rockford, IL) following the vendor's instructions. .. The immunotoxin is recovered after overnight dialysis against PBS and stored in aliquots at −80° C. Protein concentrations were determined by microtiter plate assay on a SpectraMax 340 spectrophotometer (Molecular Devices, Sunnyvale, CA) at 595 nm using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).

    Produced:

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: .. The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. Purification of the synthesized ILO-PC was performed on an SPE cartridge (SupelClean LC18, Sigma-Aldrich, St. Louis, MO) using a gradient of methanol in water.

    Concentration Assay:

    Article Title: Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
    Article Snippet: After resuspension, 3.3 μL of 300 μmol/L 5′-amine-modified EraCode DNA oligonucleotide and 5.0 μL of freshly prepared 10 g/L EDC (Pierce Chemical) were added. .. EraCoded microspheres were combined and diluted in bead buffer to generate a 50-code mixture at a concentration of 800 microspheres/μL for each class of microsphere.

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer. .. Cross-linking was quenched with the addition of β-mercaptoethanol (final concentration 20 mM, Sigma) and 4× sample buffer.

    Article Title: Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex *
    Article Snippet: .. In the case of cross-linking using EDC (Pierce), the sample was equilibrated and eluted in EDC cross-linking buffer (20 m m MES, pH 6.5, 500 m m NaCl, 2 m m MgCl2 , 0.1% CHAPS, 1 m m DTT); EDC was added to the sample to yield a final concentration of 25 m m , and N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) was added to yield a final concentration of 0.5 m m ( i.e. 2% molar ratio with respect to EDC). .. After the incubation, Tris-HCl was added to a final concentration of 50 m m and pH 8.0, and β-mercaptoethanol was added to a final concentration of 20 m m ; the sample was then incubated at 25 °C for 15 min and 750 rpm of agitation to quench the reaction.

    Thin Layer Chromatography:

    Article Title: Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium
    Article Snippet: The latter was produced from iloprost (Cayman Chemicals, Ann Arbor, MI) and N-hydroxysuccinimide (Sigma-Aldrich), using EDC (Pierce) as a coupling agent . .. The purity of the synthesized product was checked by TLC on Kieselgel 60 plates in chloroform-methanol-water (100:50:10, v/v/v) by staining with 10% CuSO4/8.5% H3 PO4 and followed by heating to 120 °C as well as by mass-spectrometry using Sciex 6500 QTRAP triple quadrupole ion trap hybrid mass spectrometer interfaced with Agilent 1290 UHPLC system.

    Fractionation:

    Article Title: A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product
    Article Snippet: Paragraph title: Fractionation of flagella and biochemical analyses ... Chemical cross-linking was performed by treating axoneme samples with EDC (Pierce Chemical Co.) for 1 h at room temperature ( ).

    Lysis:

    Article Title: Selectivity in subunit composition of Ena/VASP tetramers
    Article Snippet: .. Following a 10-min incubation on ice and centrifugation (14,000 rpm, 10 min, 4°C), EDC (final concentration 100mM, Pierce) and NHS-ester (final concentration 10mM, Thermo-Scientific) were added to the lysates and incubated at room temperature for 1 h. Untreated lysates were incubated in lysis buffer. .. Cross-linking was quenched with the addition of β-mercaptoethanol (final concentration 20 mM, Sigma) and 4× sample buffer.

    Hood:

    Article Title: Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration
    Article Snippet: The collagen completely dried under a laminar flow hood overnight and rinsed several times with MilliQ water afterwards. .. The collagen grafts then received a cross-linking treatment of 50 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, EDC, (Pierce) in 100% ethanol, agitated overnight at room temperature.

    Luminex:

    Article Title: A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity
    Article Snippet: Paragraph title: Protein coupling to Luminex beads ... 10 μL 50 mg/ml Sulfo-NHS (Pierce) and 10 μL 50 mg/ml EDC (Pierce) were added and incubated at 25 °C for 20 min while shaking.

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