propargyl maleimide  (Jena Bioscience)


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    Name:
    Propargyl Maleimide
    Description:

    Catalog Number:
    CLK-TA113-100
    Price:
    218.3
    Category:
    Click Chemistry
    Size:
    100 mg
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    Structured Review

    Jena Bioscience propargyl maleimide
    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of <t>propargyl-maleimide</t> concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

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    Images

    1) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    2) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    3) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    4) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    5) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    6) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    7) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    8) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    9) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    10) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    11) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    12) Product Images from "Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins"

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Figure Legend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Techniques Used: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.
    Figure Legend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Techniques Used: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    Related Articles

    other:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: Also, we found that preventing carry-over of excess propargyl-maleimide to the Click-labelling step was critical, by applying samples to spin columns, since any excess propargyl-maleimide may react with free azide-PEGs and therefore impair Click-PEGylation (data not shown, see the Troubleshooting Guide in ).

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: For Click-PEGred ( B), reduced thiol groups within a protein sample are reacted with a maleimide derivative, propargyl-maleimide, which contains an alkyne group.

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column.

    Concentration Assay:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: This is consistent with previous findings for redox proteomic sample preparation , and indicates the need to optimise the ratio of protein thiol content to tag compound in order to ensure complete labelling and therefore an accurate representation of protein redox status. .. Next, we optimised the concentration of propargyl-maleimide during the initial labelling of redox-reactive free thiols. .. While an excess of propargyl-maleimide at this step is desirable to ensure complete labelling, we observed that an excessive maleimide to thiol ratio led to non-specific protein labelling ( B-C), which is corroborated by previous studies , .

    Incubation:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: .. Samples were incubated with 5 mM propargyl-maleimide (Jena Bioscience CLK-TA113 or Click Chemistry Tools TA113) at 37 °C for 30 min with agitation (1400 rpm). ..

    Purification:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: .. First, the Click-PEGred protocol, designed to facilitate the selective labelling and shifting of reduced protein thiols, was tested on purified GAPDH reacted with propargyl-maleimide and derivatised with azide-PEG5000 , then visualised by Coomassie staining after SDS-PAGE ( A). .. We compared untreated GAPDH (i.e. reflecting the endogenous redox state), with GAPDH that was either fully reduced or oxidised in vitro to highlight the redox range extremes.

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: .. 3.3 Effect of azide-PEG size on redox-dependent band shifts To test the effect of azide-PEG polymer size, we compared the Click-PEGylation response of purified GAPDH reacted with propargyl-maleimide and then conjugated to a range of PEG moieties including 1, 2, and 5 kDa ( E). .. Using samples of in vitro reduced GAPDH to focus on maximal shifting, the densitometry profile plots illustrate how varying the PEG size results in a corresponding shifting of the bands, with 5 kDa (azide-PEG5000 ) providing the best band separation and resolution for the Click-PEGylation of GAPDH.

    Staining:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: .. First, the Click-PEGred protocol, designed to facilitate the selective labelling and shifting of reduced protein thiols, was tested on purified GAPDH reacted with propargyl-maleimide and derivatised with azide-PEG5000 , then visualised by Coomassie staining after SDS-PAGE ( A). .. We compared untreated GAPDH (i.e. reflecting the endogenous redox state), with GAPDH that was either fully reduced or oxidised in vitro to highlight the redox range extremes.

    SDS Page:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: .. First, the Click-PEGred protocol, designed to facilitate the selective labelling and shifting of reduced protein thiols, was tested on purified GAPDH reacted with propargyl-maleimide and derivatised with azide-PEG5000 , then visualised by Coomassie staining after SDS-PAGE ( A). .. We compared untreated GAPDH (i.e. reflecting the endogenous redox state), with GAPDH that was either fully reduced or oxidised in vitro to highlight the redox range extremes.

    Centrifugation:

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins
    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column. .. 2.1.3 Click-reaction To remove chelating agents from the above Click-PEGred and Click-PEGox samples that would impede the copper-catalysed Click reaction between propargyl-maleimide and azide-PEG, the protein was precipitated by adding 4 volumes of ice-cold acetone and storing the protein samples at –20 °C for a minimum of 2 h. Precipitated protein was pelleted by centrifugation for 30 min at 16,000×g at 4 °C and washed once with ice-cold acetone. ..

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    Jena Bioscience propargyl maleimide
    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of <t>propargyl-maleimide</t> concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
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    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Journal: Free Radical Biology & Medicine

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Figure Lengend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column.

    Techniques: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Journal: Free Radical Biology & Medicine

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Figure Lengend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column.

    Techniques: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay