Structured Review

Thermo Fisher sdab 1a6
Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs <t>1A6</t> and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same <t>sdAb-based</t> ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P
Sdab 1a6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Characterization of plasma labile heme in hemolytic conditions

Journal: The FEBS journal

doi: 10.1111/febs.14192

Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P
Figure Legend Snippet: Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

Techniques Used: Mouse Assay, Concentration Assay, Reporter Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Staining, SDS Page, Molecular Weight, Marker

Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.
Figure Legend Snippet: Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.

Techniques Used: Selection, Binding Assay, Software, Infection, Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Purification, Staining, Western Blot, DNA Sequencing, SPR Assay

Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.
Figure Legend Snippet: Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

Techniques Used: Binding Assay, Western Blot, Staining, Modification

Related Articles

Enzyme-linked Immunosorbent Assay:

Article Title: Characterization of plasma labile heme in hemolytic conditions
Article Snippet: Paragraph title: ELISA ... To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

Incubation:

Article Title: Characterization of plasma labile heme in hemolytic conditions
Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific). .. Plates were incubated with hemin (0.15−5 μm in PBS), used as standard or with plasma (1 h 30 min, RT).

Blocking Assay:

Article Title: Characterization of plasma labile heme in hemolytic conditions
Article Snippet: .. To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific). .. Plates were incubated with hemin (0.15−5 μm in PBS), used as standard or with plasma (1 h 30 min, RT).

Labeling:

Article Title: Characterization of plasma labile heme in hemolytic conditions
Article Snippet: Plates were washed (5× PBS, 0.1% Tween 20) and the rat anti-HA mAb was detected using an alkaline phosphatase labeled rabbit antiwhole rat IgG (Sigma) polyclonal Ab (1 h, RT, 1/2000) in protein-free blocking buffer. .. To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

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    Thermo Fisher sdab 1a6
    Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs <t>1A6</t> and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same <t>sdAb-based</t> ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P
    Sdab 1a6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sdab 1a6/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sdab 1a6 - by Bioz Stars, 2020-04
    92/100 stars
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    Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Mouse Assay, Concentration Assay, Reporter Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Staining, SDS Page, Molecular Weight, Marker

    Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Selection, Binding Assay, Software, Infection, Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Purification, Staining, Western Blot, DNA Sequencing, SPR Assay

    Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Binding Assay, Western Blot, Staining, Modification