Structured Review

GE Healthcare irc15p size exclusion chromatography
Redox potential determination of <t>Irc15p</t> in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).
Irc15p Size Exclusion Chromatography, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/irc15p size exclusion chromatography/product/GE Healthcare
Average 93 stars, based on 3 article reviews
Price from $9.99 to $1999.99
irc15p size exclusion chromatography - by Bioz Stars, 2020-08
93/100 stars

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1) Product Images from "Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae"

Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.3517

Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).
Figure Legend Snippet: Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

Techniques Used: Concentration Assay

Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .
Figure Legend Snippet: Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .

Techniques Used: Software

UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).
Figure Legend Snippet: UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

Techniques Used: Spectroscopy, Purification, Irradiation, Centrifugation

Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.
Figure Legend Snippet: Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.

Techniques Used: Sequencing

Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.
Figure Legend Snippet: Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.

Techniques Used: Flow Cytometry

Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.
Figure Legend Snippet: Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

Techniques Used: SDS Page, Marker, Incubation

Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.
Figure Legend Snippet: Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

Techniques Used: SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography

2) Product Images from "Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae"

Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.3517

Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).
Figure Legend Snippet: Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

Techniques Used: Concentration Assay

UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).
Figure Legend Snippet: UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

Techniques Used: Spectroscopy, Purification, Irradiation, Centrifugation

Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.
Figure Legend Snippet: Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

Techniques Used: SDS Page, Marker, Incubation

Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.
Figure Legend Snippet: Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

Techniques Used: SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography

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Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae
Article Snippet: .. To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed. ..

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    GE Healthcare irc15p size exclusion chromatography
    Redox potential determination of <t>Irc15p</t> in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).
    Irc15p Size Exclusion Chromatography, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irc15p size exclusion chromatography/product/GE Healthcare
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    irc15p size exclusion chromatography - by Bioz Stars, 2020-08
    93/100 stars
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    Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Concentration Assay

    Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Software

    UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Spectroscopy, Purification, Irradiation, Centrifugation

    Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Sequencing

    Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Flow Cytometry

    Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Marker, Incubation

    Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography

    Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Concentration Assay

    UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Spectroscopy, Purification, Irradiation, Centrifugation

    Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Marker, Incubation

    Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography