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Bio-Rad chemidoc xrs
Effects of H 2 O 2 exposure on the T. bernacchii 26S proteasome ( A ) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H 2 O 2 . Purified 26S proteasome was exposed to increasing H 2 O 2 concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H 2 O 2 treatment and native-PAGE are described in Materials and Methods section. ( B, left panel ) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; ( B, right panel ) Unoxidized and oxidized BSA were incubated with H 2 O 2 -treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. ( C ) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with <t>ChemiDoc</t> <t>XRS</t> and Quantity One software. Oxidized BSA levels after incubation with 26S and H 2 O 2 -treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H 2 O 2 -treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.
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1) Product Images from "Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii"

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii

Journal: Bioscience Reports

doi: 10.1042/BSR20160022

Effects of H 2 O 2 exposure on the T. bernacchii 26S proteasome ( A ) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H 2 O 2 . Purified 26S proteasome was exposed to increasing H 2 O 2 concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H 2 O 2 treatment and native-PAGE are described in Materials and Methods section. ( B, left panel ) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; ( B, right panel ) Unoxidized and oxidized BSA were incubated with H 2 O 2 -treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. ( C ) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with ChemiDoc XRS and Quantity One software. Oxidized BSA levels after incubation with 26S and H 2 O 2 -treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H 2 O 2 -treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.
Figure Legend Snippet: Effects of H 2 O 2 exposure on the T. bernacchii 26S proteasome ( A ) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H 2 O 2 . Purified 26S proteasome was exposed to increasing H 2 O 2 concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H 2 O 2 treatment and native-PAGE are described in Materials and Methods section. ( B, left panel ) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; ( B, right panel ) Unoxidized and oxidized BSA were incubated with H 2 O 2 -treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. ( C ) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with ChemiDoc XRS and Quantity One software. Oxidized BSA levels after incubation with 26S and H 2 O 2 -treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H 2 O 2 -treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.

Techniques Used: Clear Native PAGE, Purification, Staining, Incubation, SDS Page, Software

2) Product Images from "Therapeutic Hypothermia Activates the Endothelin and Nitric Oxide Systems after Cardiac Arrest in a Pig Model of Cardiopulmonary Resuscitation"

Article Title: Therapeutic Hypothermia Activates the Endothelin and Nitric Oxide Systems after Cardiac Arrest in a Pig Model of Cardiopulmonary Resuscitation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064792

Western Blot analysis of nNOS and eNOS. Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 4–20% SDS-acrylamide gel. Bands for neuronal NOS (nNOS) (A), endothelial NOS (eNOS) (C) and β-actin (A and C) were detected at 160 kDa, 135 kDa and 42 kDa, respectively. Protein band intensities for nNOS (B) and eNOS (D) were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin). Error bars represent standard error of the mean (SEM). Significant results (p
Figure Legend Snippet: Western Blot analysis of nNOS and eNOS. Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 4–20% SDS-acrylamide gel. Bands for neuronal NOS (nNOS) (A), endothelial NOS (eNOS) (C) and β-actin (A and C) were detected at 160 kDa, 135 kDa and 42 kDa, respectively. Protein band intensities for nNOS (B) and eNOS (D) were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin). Error bars represent standard error of the mean (SEM). Significant results (p

Techniques Used: Western Blot, Acrylamide Gel Assay, Software

Western Blot analysis of ETAR and ETBR. Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 12% SDS-acrylamide gel. Bands for endothelin A receptor (ETAR), endothelin B receptor (ETBR) and β-actin were detected at 25 kDa, 50 kDa and 42 kDa, respectively (A). Protein band intensities were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin) (B). Error bars represent standard error of the mean (SEM). Significant results (p
Figure Legend Snippet: Western Blot analysis of ETAR and ETBR. Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 12% SDS-acrylamide gel. Bands for endothelin A receptor (ETAR), endothelin B receptor (ETBR) and β-actin were detected at 25 kDa, 50 kDa and 42 kDa, respectively (A). Protein band intensities were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin) (B). Error bars represent standard error of the mean (SEM). Significant results (p

Techniques Used: Western Blot, Acrylamide Gel Assay, Software

3) Product Images from "Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii"

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii

Journal: Bioscience Reports

doi: 10.1042/BSR20160022

Effects of H 2 O 2 exposure on the T. bernacchii 26S proteasome ( A ) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H 2 O 2 . Purified 26S proteasome was exposed to increasing H 2 O 2 concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H 2 O 2 treatment and native-PAGE are described in Materials and Methods section. ( B, left panel ) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; ( B, right panel ) Unoxidized and oxidized BSA were incubated with H 2 O 2 -treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. ( C ) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with ChemiDoc XRS and Quantity One software. Oxidized BSA levels after incubation with 26S and H 2 O 2 -treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H 2 O 2 -treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.
Figure Legend Snippet: Effects of H 2 O 2 exposure on the T. bernacchii 26S proteasome ( A ) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H 2 O 2 . Purified 26S proteasome was exposed to increasing H 2 O 2 concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H 2 O 2 treatment and native-PAGE are described in Materials and Methods section. ( B, left panel ) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; ( B, right panel ) Unoxidized and oxidized BSA were incubated with H 2 O 2 -treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. ( C ) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with ChemiDoc XRS and Quantity One software. Oxidized BSA levels after incubation with 26S and H 2 O 2 -treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H 2 O 2 -treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.

Techniques Used: Clear Native PAGE, Purification, Staining, Incubation, SDS Page, Software

4) Product Images from "Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons"

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2014.00246

Effect of glutamate on mRNA and protein expression of AQP9, MCT2, and LDHA in cultured rat hippocampal neurons (A) Primary cultures of rat hippocampal neurons were treated with glutamate 100 μM for 1 h . Then mRNA was extracted and analyzed for AQP9, MCT2 and LDH mRNA expression using RT-PCR. Results are expressed as relative expression vs. control with mean value for control set at 1 after values had been normalized using β-actin as internal reference. Results represent mean ± SD with n = 3. The experiment was repeated three times from different cultures with similar results. Statistical analysis was performed using a non-parametric Mann–Whitney test. No significant difference was observed. (B) Primary cultures of rat hippocampal neurons were treated with glutamate 100 μM for 1 h. Then proteins were extracted and analyzed for AQP9, MCT2, and LDH protein expression using Western blot. Blot pictures were obtained with Bio-Rad Chemidoc™ XRS system (BioRad, Cressier, Switzerland) and quantified using ImageJ software. Quantitative results are expressed as percentage of control after the values had been normalized using β-tubulin signal as reference. Results represent mean ± SD with n = 3. The experiment was repeated three times from different cultures with similar results. Statistical analysis was performed using a non-parametric Mann–Whitney test. An asterisk indicates protein level significantly different from control with p
Figure Legend Snippet: Effect of glutamate on mRNA and protein expression of AQP9, MCT2, and LDHA in cultured rat hippocampal neurons (A) Primary cultures of rat hippocampal neurons were treated with glutamate 100 μM for 1 h . Then mRNA was extracted and analyzed for AQP9, MCT2 and LDH mRNA expression using RT-PCR. Results are expressed as relative expression vs. control with mean value for control set at 1 after values had been normalized using β-actin as internal reference. Results represent mean ± SD with n = 3. The experiment was repeated three times from different cultures with similar results. Statistical analysis was performed using a non-parametric Mann–Whitney test. No significant difference was observed. (B) Primary cultures of rat hippocampal neurons were treated with glutamate 100 μM for 1 h. Then proteins were extracted and analyzed for AQP9, MCT2, and LDH protein expression using Western blot. Blot pictures were obtained with Bio-Rad Chemidoc™ XRS system (BioRad, Cressier, Switzerland) and quantified using ImageJ software. Quantitative results are expressed as percentage of control after the values had been normalized using β-tubulin signal as reference. Results represent mean ± SD with n = 3. The experiment was repeated three times from different cultures with similar results. Statistical analysis was performed using a non-parametric Mann–Whitney test. An asterisk indicates protein level significantly different from control with p

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Western Blot, Software

5) Product Images from "Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages"

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

Journal: BioMed Research International

doi: 10.1155/2014/901617

Stability of natural and modified siRNAs in 100% fetal bovine serum at 37°C. After incubation, siRNAs were analyzed by PAGE and ethidium bromide staining. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA bands were quantified by Image Lab software (Bio-Rad). Signal intensity values at t 0 were set at 1.
Figure Legend Snippet: Stability of natural and modified siRNAs in 100% fetal bovine serum at 37°C. After incubation, siRNAs were analyzed by PAGE and ethidium bromide staining. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA bands were quantified by Image Lab software (Bio-Rad). Signal intensity values at t 0 were set at 1.

Techniques Used: Modification, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Software

6) Product Images from "The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages"

Article Title: The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

Journal: Biochemical Journal

doi: 10.1042/BJ20100082

Effect of HOSCN on the extent of protein phosphorylation in J774A.1 cells ( A ) MAPK array for phosphorylated proteins detected in J774A.1 cells (1×10 6 ) treated with either (a) PBS or (b) 50 μM HOSCN in PBS, pH 7.4, for 15 min at 37 °C. After oxidant (or control) exposure, cells were lysed using the buffer provided with the kit, and 300 μg of protein (determined using the Bio-Rad protein assay) used for each array. Following binding, the arrays were washed and reacted with the supplied antibody cocktail as specified by the manufacturer. Chemiluminescent detection was performed with ECL with the signals detected using a Bio-Rad ChemiDoc XRS system and Bio-Rad Quantity One software (version 4.6.1). Pairs of protein spots marked ‘1’ are positive controls. Duplicate spots from phosphorylated ERK2 (MAPK1; spots marked ‘2’) and p38α (spots marked ‘3’) are indicated. ( B ) Western blots of total and phosphorylated p38α, and corresponding gel densitometry (ratio of phosphorylated to total, n =3 independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). ( C ) Western blots of total and phosphorylated MKK3 (solid bars) and MKK6 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). ( D ) Western blots of total and phosphorylated ERK1 (solid bars) and ERK2 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). Statistical analyses compared the HOSCN-treated samples with the untreated control (PBS-treated) cells using one-way ANOVA with Bonferroni's post-hoc test, * P
Figure Legend Snippet: Effect of HOSCN on the extent of protein phosphorylation in J774A.1 cells ( A ) MAPK array for phosphorylated proteins detected in J774A.1 cells (1×10 6 ) treated with either (a) PBS or (b) 50 μM HOSCN in PBS, pH 7.4, for 15 min at 37 °C. After oxidant (or control) exposure, cells were lysed using the buffer provided with the kit, and 300 μg of protein (determined using the Bio-Rad protein assay) used for each array. Following binding, the arrays were washed and reacted with the supplied antibody cocktail as specified by the manufacturer. Chemiluminescent detection was performed with ECL with the signals detected using a Bio-Rad ChemiDoc XRS system and Bio-Rad Quantity One software (version 4.6.1). Pairs of protein spots marked ‘1’ are positive controls. Duplicate spots from phosphorylated ERK2 (MAPK1; spots marked ‘2’) and p38α (spots marked ‘3’) are indicated. ( B ) Western blots of total and phosphorylated p38α, and corresponding gel densitometry (ratio of phosphorylated to total, n =3 independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). ( C ) Western blots of total and phosphorylated MKK3 (solid bars) and MKK6 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). ( D ) Western blots of total and phosphorylated ERK1 (solid bars) and ERK2 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for ( A ). Statistical analyses compared the HOSCN-treated samples with the untreated control (PBS-treated) cells using one-way ANOVA with Bonferroni's post-hoc test, * P

Techniques Used: Binding Assay, Software, Western Blot

7) Product Images from "Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly"

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-8-9

Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.
Figure Legend Snippet: Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Incubation, Isolation, Western Blot, Software

8) Product Images from "Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine"

Article Title: Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17040577

Inactivation of p38 and Erk1/2 in the low concentration of BBR-treated HCC cells by western blot. SMMC-7721 and Bel-7402 cells were treated with BBR for 6 h. Proteins were collected, determined and expression of p-p38, p-Erk1/2, p-JNK/SAPK and p-Src were analyzed by western blot. The expression of total proteins was used as internal standard. ( A ) Protein expression was observed by ChemiDoc™ XRS + Molecular Imager; ( B ) Protein expression was calculated by ImageJ 1.38x software. * p
Figure Legend Snippet: Inactivation of p38 and Erk1/2 in the low concentration of BBR-treated HCC cells by western blot. SMMC-7721 and Bel-7402 cells were treated with BBR for 6 h. Proteins were collected, determined and expression of p-p38, p-Erk1/2, p-JNK/SAPK and p-Src were analyzed by western blot. The expression of total proteins was used as internal standard. ( A ) Protein expression was observed by ChemiDoc™ XRS + Molecular Imager; ( B ) Protein expression was calculated by ImageJ 1.38x software. * p

Techniques Used: Concentration Assay, Western Blot, Expressing, Software

Induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of urokinase-type plasminogen activator (uPA) in BBR-treated HCC cells by western blot. SMMC-7721 and Bel-7402 cells were treated with BBR for 6 h. Proteins were collected, determined and expression of PAI-1, TIMP-1, TIMP-2, uPA and tissue-type plasminogen activator (tPA) were analyzed by western blot. β-actin was used as internal standard. ( A ) Protein expression was observed by ChemiDoc™ XRS + Molecular Imager; ( B ) Protein expression was calculated by ImageJ 1.38x software. * p
Figure Legend Snippet: Induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of urokinase-type plasminogen activator (uPA) in BBR-treated HCC cells by western blot. SMMC-7721 and Bel-7402 cells were treated with BBR for 6 h. Proteins were collected, determined and expression of PAI-1, TIMP-1, TIMP-2, uPA and tissue-type plasminogen activator (tPA) were analyzed by western blot. β-actin was used as internal standard. ( A ) Protein expression was observed by ChemiDoc™ XRS + Molecular Imager; ( B ) Protein expression was calculated by ImageJ 1.38x software. * p

Techniques Used: Inhibition, Western Blot, Expressing, Software

9) Product Images from "Sialidase Unmasks Mucin Domain Epitopes of Lubricin"

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin

Journal: Journal of Histochemistry and Cytochemistry

doi: 10.1369/0022155416668139

Sialidase enhances the reactivity of the S6.79 monoclonal antibody in human body fluids. S6.79 Western blot analysis of normal human body fluids (SF, plasma, serum, and eye sleep extract) treated with sialidase or buffer control for 18 hr (6.390-sec exposure; ChemiDoc XRS+; Bio-Rad). Abbreviation: SF, synovial fluid.
Figure Legend Snippet: Sialidase enhances the reactivity of the S6.79 monoclonal antibody in human body fluids. S6.79 Western blot analysis of normal human body fluids (SF, plasma, serum, and eye sleep extract) treated with sialidase or buffer control for 18 hr (6.390-sec exposure; ChemiDoc XRS+; Bio-Rad). Abbreviation: SF, synovial fluid.

Techniques Used: Western Blot, Size-exclusion Chromatography

10) Product Images from "Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink"

Article Title: Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky759

SNM1A can generate a nick to initiate trimming to the crosslink. ( A ) DNA substrate design mimicking stalled replication fork. ( B ) Substrate end modifications and corresponding nuclease activities monitored. F, fluorophore; OH, hydroxyl; P, phosphate. ( C ) In vitro analysis of SNM1A nuclease activities on 5′flap crosslinked DNA. SNM1A (0.4 μM) was incubated with substrate (0.1μM) as shown in (A) with or without an SJG-136 crosslink. Substrates are labeled as indicated in (B). Reactions were stopped after 0, 2 and 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( D ) Summary of nuclease events on 5′ flap substrate with or without an SJG crosslink. Blue arrows indicate initial endonuclease cleavage. Dark gray arrows indicate exonuclease product. Light grey arrow indicates translesional nuclease product. ( E ) Quantification of product formed in the absence of the ICL, at the ICL, or past the ICL. Products were quantified using ImageLab.
Figure Legend Snippet: SNM1A can generate a nick to initiate trimming to the crosslink. ( A ) DNA substrate design mimicking stalled replication fork. ( B ) Substrate end modifications and corresponding nuclease activities monitored. F, fluorophore; OH, hydroxyl; P, phosphate. ( C ) In vitro analysis of SNM1A nuclease activities on 5′flap crosslinked DNA. SNM1A (0.4 μM) was incubated with substrate (0.1μM) as shown in (A) with or without an SJG-136 crosslink. Substrates are labeled as indicated in (B). Reactions were stopped after 0, 2 and 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( D ) Summary of nuclease events on 5′ flap substrate with or without an SJG crosslink. Blue arrows indicate initial endonuclease cleavage. Dark gray arrows indicate exonuclease product. Light grey arrow indicates translesional nuclease product. ( E ) Quantification of product formed in the absence of the ICL, at the ICL, or past the ICL. Products were quantified using ImageLab.

Techniques Used: In Vitro, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

Stability of SJG-136 crosslinks are DNA length-dependent. Short ( A ) and long ( B ) ICL substrates with 3′ fluorescent label. In vitro analysis of SNM1A translesional nuclease activity on short ICL ( C ) or long ICL ( D ). SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with duplex DNA substrate (0.2 μM) with (left and middle; lanes 1–8) or without (right; lanes 9–12) an SJG-136 crosslink. Reactions were stopped after 0, 5 and 60 min. Crosslinked products were heat denatured (shown in middle panels) to disrupt the crosslink and allow visualization of the top strand alone. A schematic of products is shown with the dark strand represents the labelled DNA visible on the gel. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. * denotes residual crosslinked DNA following heat treatment. ** denotes unphosphorylated substrate.
Figure Legend Snippet: Stability of SJG-136 crosslinks are DNA length-dependent. Short ( A ) and long ( B ) ICL substrates with 3′ fluorescent label. In vitro analysis of SNM1A translesional nuclease activity on short ICL ( C ) or long ICL ( D ). SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with duplex DNA substrate (0.2 μM) with (left and middle; lanes 1–8) or without (right; lanes 9–12) an SJG-136 crosslink. Reactions were stopped after 0, 5 and 60 min. Crosslinked products were heat denatured (shown in middle panels) to disrupt the crosslink and allow visualization of the top strand alone. A schematic of products is shown with the dark strand represents the labelled DNA visible on the gel. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. * denotes residual crosslinked DNA following heat treatment. ** denotes unphosphorylated substrate.

Techniques Used: In Vitro, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis

SNM1A has single-strand specific endonuclease activity. ( A ) In vitro time course analysis of SNM1A nuclease activity on 5′ fluorescently-labeled ssDNA. SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with indicated ssDNA (0.1 μM). Reactions were stopped after 0, 2 or 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( B ).
Figure Legend Snippet: SNM1A has single-strand specific endonuclease activity. ( A ) In vitro time course analysis of SNM1A nuclease activity on 5′ fluorescently-labeled ssDNA. SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with indicated ssDNA (0.1 μM). Reactions were stopped after 0, 2 or 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( B ).

Techniques Used: Activity Assay, In Vitro, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

11) Product Images from "HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo"

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo

Journal: Retrovirology

doi: 10.1186/s12977-019-0507-9

Construction of HTLV-1 proviral clones. Site-directed mutagenesis was utilized to abrogate CTCF binding. a Alignment of the consensus CTCF-binding sequence with HTLV-1, HTLV-1p12Stop, and HTLV-1∆CTCF in the context of accessory gene p12. HTLV-1∆CTCF contains mutations that abolish CTCF binding (blue). These mutations disrupt the p12 reading frame, therefore a stop mutation (red) that truncates p12 by 23 amino acids was introduced immediately upstream. HTLV-1p12Stop serves as a control by containing only the aforementioned stop codon (red). b Abolition of CTCF binding was confirmed via electrophoretic mobility shift assay. EMSA was performed using the Light Chemiluminescent EMSA kit (Thermo Scientific) and following the manufacturer’s protocol with some modification. Briefly, nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in the presence or absence of the CTCF antibody. Protein bound DNA was separated from unbound DNA in a polyacrylamide gel and transferred to a nylon membrane. DNA was then cross-linked to the membrane. The membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer and then exposed to the substrate solution. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad)
Figure Legend Snippet: Construction of HTLV-1 proviral clones. Site-directed mutagenesis was utilized to abrogate CTCF binding. a Alignment of the consensus CTCF-binding sequence with HTLV-1, HTLV-1p12Stop, and HTLV-1∆CTCF in the context of accessory gene p12. HTLV-1∆CTCF contains mutations that abolish CTCF binding (blue). These mutations disrupt the p12 reading frame, therefore a stop mutation (red) that truncates p12 by 23 amino acids was introduced immediately upstream. HTLV-1p12Stop serves as a control by containing only the aforementioned stop codon (red). b Abolition of CTCF binding was confirmed via electrophoretic mobility shift assay. EMSA was performed using the Light Chemiluminescent EMSA kit (Thermo Scientific) and following the manufacturer’s protocol with some modification. Briefly, nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in the presence or absence of the CTCF antibody. Protein bound DNA was separated from unbound DNA in a polyacrylamide gel and transferred to a nylon membrane. DNA was then cross-linked to the membrane. The membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer and then exposed to the substrate solution. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad)

Techniques Used: Clone Assay, Mutagenesis, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Modification, Transfection, Plasmid Preparation, Incubation, Labeling, Blocking Assay

12) Product Images from "A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets"

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets

Journal: Journal of Proteomics

doi: 10.1016/j.jprot.2013.11.011

Histone H3 is a TG2 substrate during FPIS in INS-1E and in human pancreatic islets. 70 μg of INS-1E proteins taken from the nuclear/mitochondrial enriched fraction were first resolved using classical SDS-PAGE. 15% acrylamide gel 7 × 7cm and then electrotransferred. Cells labeled with A25 peptide. Images collected using ChemiDoc XRS + exposing for 60 s. A: Total biotinylated INS-1E nuclear/mitochondrial proteins. Western blot performed on the total lysate of nuclear/mitochondrial fraction revealed using streptavidin–HPR; 1: Resting cells. 2: Cells stimulated for 2 min with 15.5 mM glucose. B: Purified biotinylated proteins from INS-1E cells. 1: Resting cells. 2: Cells stimulated for 2 min with 15.5 mM glucose. C: Purified biotinylated proteins from human pancreatic islets. 1: Human pancreatic islets grown in 5.5 mM glucose. 2: Human pancreatic islets stimulated 3 min with 11 mM glucose. In B and C the Western blot was revealed using rabbit polyclonal antiserum against histone H3. Arrows indicate bands corresponding to histone H3 at the expected MW according to the manufacturer. In C lower bands corresponding to histone H3 are over exposed and for this reason appear white.
Figure Legend Snippet: Histone H3 is a TG2 substrate during FPIS in INS-1E and in human pancreatic islets. 70 μg of INS-1E proteins taken from the nuclear/mitochondrial enriched fraction were first resolved using classical SDS-PAGE. 15% acrylamide gel 7 × 7cm and then electrotransferred. Cells labeled with A25 peptide. Images collected using ChemiDoc XRS + exposing for 60 s. A: Total biotinylated INS-1E nuclear/mitochondrial proteins. Western blot performed on the total lysate of nuclear/mitochondrial fraction revealed using streptavidin–HPR; 1: Resting cells. 2: Cells stimulated for 2 min with 15.5 mM glucose. B: Purified biotinylated proteins from INS-1E cells. 1: Resting cells. 2: Cells stimulated for 2 min with 15.5 mM glucose. C: Purified biotinylated proteins from human pancreatic islets. 1: Human pancreatic islets grown in 5.5 mM glucose. 2: Human pancreatic islets stimulated 3 min with 11 mM glucose. In B and C the Western blot was revealed using rabbit polyclonal antiserum against histone H3. Arrows indicate bands corresponding to histone H3 at the expected MW according to the manufacturer. In C lower bands corresponding to histone H3 are over exposed and for this reason appear white.

Techniques Used: SDS Page, Acrylamide Gel Assay, Labeling, Western Blot, Purification

TG2 localizes in nuclear/mitochondrial enriched fraction. INS-1E proteins from the nuclear/mitochondrial (A, C, E and G) and cytoplasmic (B, D, F and H) enriched fractions were resolved by 2D-electrophoresis and transferred to nitrocellulose for Western blot analysis. TG2 segregation in the subcellular fractions was inferred by Western blot using a rabbit polyclonal serum anti TG2 (panels A and B). The quality of the fraction enrichment was assessed by using: a mouse monoclonal anti ATP synthase subunit alpha as marker for mitochondria (panels C and D); a rabbit polyclonal serum anti GAPDH as cytoplasmic marker (panels E and F); and a goat polyclonal serum anti LaminA/C as nuclear marker (panels G and H). Reactivities were revealed using specific HRP-conjugated secondary antibodies and chemiluminescence reaction. Blot images were collected using ChemiDoc XRS + exposure time 30 s.
Figure Legend Snippet: TG2 localizes in nuclear/mitochondrial enriched fraction. INS-1E proteins from the nuclear/mitochondrial (A, C, E and G) and cytoplasmic (B, D, F and H) enriched fractions were resolved by 2D-electrophoresis and transferred to nitrocellulose for Western blot analysis. TG2 segregation in the subcellular fractions was inferred by Western blot using a rabbit polyclonal serum anti TG2 (panels A and B). The quality of the fraction enrichment was assessed by using: a mouse monoclonal anti ATP synthase subunit alpha as marker for mitochondria (panels C and D); a rabbit polyclonal serum anti GAPDH as cytoplasmic marker (panels E and F); and a goat polyclonal serum anti LaminA/C as nuclear marker (panels G and H). Reactivities were revealed using specific HRP-conjugated secondary antibodies and chemiluminescence reaction. Blot images were collected using ChemiDoc XRS + exposure time 30 s.

Techniques Used: Two-Dimensional Gel Electrophoresis, Western Blot, Marker

Kinetic of transamidation reaction of TG2 during FPIS. 200 μg of INS-1E proteins from the nuclear/mitochondrial enriched fraction were first resolved on 7 cm strips 3–10 NL and then electroblotted. Cells labeled with A25 peptide. Western blot was revealed using streptavidin–HPR 1/1000 in 2% BSA in TTBS followed by ECL. Blot images were collected using ChemiDoc XRS +. Exposure time: 60 s. A: INS-1E maintained at 2.5 mM glucose; B: INS-1E stimulated for 2 min; C: INS-1E stimulated for 5 min; D: INS-1E stimulated for 8 min at 15.5 mM glucose; E: INS-1E stimulated for 15 min. Stimulation performed at 15.5 mM glucose F: The graph shows the changes of Ca 2 + intracellular concentration during FIPS. Ca 2 + levels were not directly measured and were assumed to be equal to that assessed previously in this cell line [28] . G: Histogram indicates the changes in the number of spots for each condition. The number is reported as the mean and SE of three independent experiments.
Figure Legend Snippet: Kinetic of transamidation reaction of TG2 during FPIS. 200 μg of INS-1E proteins from the nuclear/mitochondrial enriched fraction were first resolved on 7 cm strips 3–10 NL and then electroblotted. Cells labeled with A25 peptide. Western blot was revealed using streptavidin–HPR 1/1000 in 2% BSA in TTBS followed by ECL. Blot images were collected using ChemiDoc XRS +. Exposure time: 60 s. A: INS-1E maintained at 2.5 mM glucose; B: INS-1E stimulated for 2 min; C: INS-1E stimulated for 5 min; D: INS-1E stimulated for 8 min at 15.5 mM glucose; E: INS-1E stimulated for 15 min. Stimulation performed at 15.5 mM glucose F: The graph shows the changes of Ca 2 + intracellular concentration during FIPS. Ca 2 + levels were not directly measured and were assumed to be equal to that assessed previously in this cell line [28] . G: Histogram indicates the changes in the number of spots for each condition. The number is reported as the mean and SE of three independent experiments.

Techniques Used: Labeling, Western Blot, Concentration Assay

13) Product Images from "Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue"

Article Title: Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034002

C/EBPα, C/EBPβ mRNA and protein expression in iWAT (A) and in the liver (C) of SD rats. Effects of central (icv) dexamethasone infusion for 2 days. A and C) Results are expressed as percent of relative mRNA expression compared to that obtained in control rats (100%). The analysis was performed in duplicate and the results were normalized with RPS29, β -actin and GAPDH expression. Mean ± SEM of n = 7–8 rats per group. B) Representative Western blots of C/EBPα, C/EBPβ and phosphorylated C/EBPβ-ser 105 (n = 5 per group). Quantification was performed using the ChemiDoc™ XRS and the Quantity One™ software. * P
Figure Legend Snippet: C/EBPα, C/EBPβ mRNA and protein expression in iWAT (A) and in the liver (C) of SD rats. Effects of central (icv) dexamethasone infusion for 2 days. A and C) Results are expressed as percent of relative mRNA expression compared to that obtained in control rats (100%). The analysis was performed in duplicate and the results were normalized with RPS29, β -actin and GAPDH expression. Mean ± SEM of n = 7–8 rats per group. B) Representative Western blots of C/EBPα, C/EBPβ and phosphorylated C/EBPβ-ser 105 (n = 5 per group). Quantification was performed using the ChemiDoc™ XRS and the Quantity One™ software. * P

Techniques Used: Expressing, Western Blot, Software

14) Product Images from "Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink"

Article Title: Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky759

SNM1A can generate a nick to initiate trimming to the crosslink. ( A ) DNA substrate design mimicking stalled replication fork. ( B ) Substrate end modifications and corresponding nuclease activities monitored. F, fluorophore; OH, hydroxyl; P, phosphate. ( C ) In vitro analysis of SNM1A nuclease activities on 5′flap crosslinked DNA. SNM1A (0.4 μM) was incubated with substrate (0.1μM) as shown in (A) with or without an SJG-136 crosslink. Substrates are labeled as indicated in (B). Reactions were stopped after 0, 2 and 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( D ) Summary of nuclease events on 5′ flap substrate with or without an SJG crosslink. Blue arrows indicate initial endonuclease cleavage. Dark gray arrows indicate exonuclease product. Light grey arrow indicates translesional nuclease product. ( E ) Quantification of product formed in the absence of the ICL, at the ICL, or past the ICL. Products were quantified using ImageLab.
Figure Legend Snippet: SNM1A can generate a nick to initiate trimming to the crosslink. ( A ) DNA substrate design mimicking stalled replication fork. ( B ) Substrate end modifications and corresponding nuclease activities monitored. F, fluorophore; OH, hydroxyl; P, phosphate. ( C ) In vitro analysis of SNM1A nuclease activities on 5′flap crosslinked DNA. SNM1A (0.4 μM) was incubated with substrate (0.1μM) as shown in (A) with or without an SJG-136 crosslink. Substrates are labeled as indicated in (B). Reactions were stopped after 0, 2 and 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( D ) Summary of nuclease events on 5′ flap substrate with or without an SJG crosslink. Blue arrows indicate initial endonuclease cleavage. Dark gray arrows indicate exonuclease product. Light grey arrow indicates translesional nuclease product. ( E ) Quantification of product formed in the absence of the ICL, at the ICL, or past the ICL. Products were quantified using ImageLab.

Techniques Used: In Vitro, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

Stability of SJG-136 crosslinks are DNA length-dependent. Short ( A ) and long ( B ) ICL substrates with 3′ fluorescent label. In vitro analysis of SNM1A translesional nuclease activity on short ICL ( C ) or long ICL ( D ). SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with duplex DNA substrate (0.2 μM) with (left and middle; lanes 1–8) or without (right; lanes 9–12) an SJG-136 crosslink. Reactions were stopped after 0, 5 and 60 min. Crosslinked products were heat denatured (shown in middle panels) to disrupt the crosslink and allow visualization of the top strand alone. A schematic of products is shown with the dark strand represents the labelled DNA visible on the gel. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. * denotes residual crosslinked DNA following heat treatment. ** denotes unphosphorylated substrate.
Figure Legend Snippet: Stability of SJG-136 crosslinks are DNA length-dependent. Short ( A ) and long ( B ) ICL substrates with 3′ fluorescent label. In vitro analysis of SNM1A translesional nuclease activity on short ICL ( C ) or long ICL ( D ). SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with duplex DNA substrate (0.2 μM) with (left and middle; lanes 1–8) or without (right; lanes 9–12) an SJG-136 crosslink. Reactions were stopped after 0, 5 and 60 min. Crosslinked products were heat denatured (shown in middle panels) to disrupt the crosslink and allow visualization of the top strand alone. A schematic of products is shown with the dark strand represents the labelled DNA visible on the gel. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. * denotes residual crosslinked DNA following heat treatment. ** denotes unphosphorylated substrate.

Techniques Used: In Vitro, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis

SNM1A has single-strand specific endonuclease activity. ( A ) In vitro time course analysis of SNM1A nuclease activity on 5′ fluorescently-labeled ssDNA. SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with indicated ssDNA (0.1 μM). Reactions were stopped after 0, 2 or 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( B ) Quantification of substrate processed at indicated time points. Substrates were quantified using ImageLab. * denotes an aberrant fluorophore-dependent product ( Supplementary 4 ).
Figure Legend Snippet: SNM1A has single-strand specific endonuclease activity. ( A ) In vitro time course analysis of SNM1A nuclease activity on 5′ fluorescently-labeled ssDNA. SNM1A (0.2 μM) or Lambda exonuclease (5 units) were incubated with indicated ssDNA (0.1 μM). Reactions were stopped after 0, 2 or 30 min. Products were resolved using 20% denaturing PAGE and imaged with the ChemiDoc XRS (BioRad) at 526 nm. ( B ) Quantification of substrate processed at indicated time points. Substrates were quantified using ImageLab. * denotes an aberrant fluorophore-dependent product ( Supplementary 4 ).

Techniques Used: Activity Assay, In Vitro, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

15) Product Images from "The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression"

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression

Journal: Medical Sciences

doi: 10.3390/medsci6010008

Immunoblot analysis of capsular polysaccharides in TIGR4 and mutant strains. All strains were cultured in THY supplemented with fetal bovine serum (FBS) to mid-log phase. Total capsular polysaccharide (CPS) isolated from equal number of cells for each strain, and 3× dilutions were spotted onto a nitrocellulose membrane. Membranes were probed with rabbit anti-serotype 4 sera and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. Membranes were developed with enhanced chemiluminiscence (ECL) detection and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA). Data from representative immunoblot from two independent colonies for each strain are shown.
Figure Legend Snippet: Immunoblot analysis of capsular polysaccharides in TIGR4 and mutant strains. All strains were cultured in THY supplemented with fetal bovine serum (FBS) to mid-log phase. Total capsular polysaccharide (CPS) isolated from equal number of cells for each strain, and 3× dilutions were spotted onto a nitrocellulose membrane. Membranes were probed with rabbit anti-serotype 4 sera and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. Membranes were developed with enhanced chemiluminiscence (ECL) detection and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA). Data from representative immunoblot from two independent colonies for each strain are shown.

Techniques Used: Mutagenesis, Cell Culture, Isolation, Software

Related Articles

Clone Assay:

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: Paragraph title: Plasmids and cloning ... Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Centrifugation:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Proteasome activity and Ub-protein levels T. bernacchii and D. labrax hemolysates were obtained from erythrocytes by incubation in hypotonic solution (25 mM Tris/HCl, pH 7.5) for 30 min on ice and centrifugation at 9200 g for 40 min at 4°C. .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Autoradiography:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories). .. Chemiluminescence was quantified using Quantity One Software (Bio-Rad Laboratories) and the acquisition data were performed under conditions to prevent saturation of the chemiluminescence signal.

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Blocking Assay:

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: For protein detection, membranes were incubated in a blocking solution of Tris-buffered saline supplemented with Tween-20 (TBST; Tris-HCl, pH 7.5, 50 mmol/L; NaCl, 150 mmol/L; and Tween-20, 0.1%) containing 5% non-fat milk for 1 h at room temperature. .. After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad).

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Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Following blocking, the membrane was incubated with Ub conjugates specific primary antibody HRP conjugate (1:2500, produced by Enzo Life Sciences) for 1 h at room temperature. .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: DNA was then cross-linked to the membrane with the UV Stratalinker 1800 (Stratagene) for 1 min. After 15 min blocking, the membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer for 15 min and then exposed to the substrate solution. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Electrophoresis:

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages
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Article Snippet: The extracts were resolved by electrophoresis on 12% SDS-acrylamide gels and transferred to Hybond-P PVDF membranes (GE Healthcare, Uppsala, Sweden). .. Protein band densities were digitally quantified and normalized to the loading control (β-actin) with a ChemiDoc XRS and Quantity One image analysis software (Bio-Rad laboratories, USA).

Article Title: Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine
Article Snippet: The electrophoresis was conducted on a SDS-PAGE gel at 120 V using Powerpac Basic electrometer (Bio-Rad, Hercules, CA, USA) following the Bio-Rad Laboratories handbook. .. The membrane was blocked by 5% BSA and incubated with the primary antibodies on a rocker overnight, followed by incubating with the secondary antibodies on a rocker for 1 h. After rinsing four times (10 min × 1 and 5 min × 3), the membrane was developed by Millipore ECL chemiluminescence kit (Millipotre Corporation, Billerica, MA, USA) and exposure in ChemiDoc™ XRS+ Molecular Imager (Bio-Rad).

Incubation:

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages
Article Snippet: Equal amounts of siRNAs before serum incubation were extracted with phenol and loaded as controls. .. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA electrophoretic bands were quantified by Image Lab software (Bio-Rad).

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Biotinylated proteins were then revealed by incubation with streptavin-HPR (1/2000 in 1% BSA in 100 mM Tris, 150 mM NaCl pH 7.5, 0.1% Tween 20, TTBS). .. Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure.

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: After washing in TBST, membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies in TBST containing 1% of non-fat milk, for 2 h at room temperature. .. After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad).

Article Title: The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages
Article Snippet: Total cell protein (300 μg) was hybridized to the array membranes, the arrays were washed and incubated with the detection antibody cocktail labelled with streptavidin–HRP. .. Signals were directly digitized using a ChemiDoc XRS (Bio-Rad).

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Article Snippet: After washing, membranes were incubated with anti-rabbit IgG-conjugated to horseradish peroxidase (1∶3000). .. Protein band densities were digitally quantified and normalized to the loading control (β-actin) with a ChemiDoc XRS and Quantity One image analysis software (Bio-Rad laboratories, USA).

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly
Article Snippet: After incubation with the primary antibody the membranes were washed twice in Tris-buffered saline (TBS) containing 0.1% Tween-20 (Sigma-Aldrich Corp.) and incubated for 1 h with sheep anti-rabbit IgG or sheep anti-mouse IgG (Amersham Biosciences, Little Calfont, UK), both conjugated with horseradish peroxidase. .. After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad).

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression
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Article Title: Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink
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Article Title: Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue
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Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
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Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: .. Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad). .. The band volume (pixel density × area) was analyzed by Image Lab software version 4.1 (Bio-Rad), and fold changes were generated by calculating the sialidase-treated lubricin band volume divided by the untreated lubricin band volume.

Article Title: Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine
Article Snippet: .. The membrane was blocked by 5% BSA and incubated with the primary antibodies on a rocker overnight, followed by incubating with the secondary antibodies on a rocker for 1 h. After rinsing four times (10 min × 1 and 5 min × 3), the membrane was developed by Millipore ECL chemiluminescence kit (Millipotre Corporation, Billerica, MA, USA) and exposure in ChemiDoc™ XRS+ Molecular Imager (Bio-Rad). .. Confocal Microscope HCC cells were treated with 50 μM of BBR for 6h and were fixed with 4% paraformaldehyde and penetrated with 0.3% Triton-X100.

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Membranes were next incubated with the piscine anti-beta 1/beta 5 (LS-C111925 rabbit IgG, 1:1000, purchased from LifeSpan BioSciences) and piscine anti-Rpt1 (LS-C290473 rabbit IgG, 1:1000, purchased from LifeSpan BioSciences) primary antibodies (1 h at room temperature) and then with the HRP-conjugated secondary antibodies (1 h at room temperature). .. Immune complexes formed were visualized by enhanced chemiluminescence and autoradiography (Amersham Biosciences) and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: DNA was then cross-linked to the membrane with the UV Stratalinker 1800 (Stratagene) for 1 min. After 15 min blocking, the membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer for 15 min and then exposed to the substrate solution. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Activity Assay:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Paragraph title: Proteasome activity and Ub-protein levels ... Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Expressing:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Immune complexes formed were visualized by enhanced chemiluminescence and autoradiography (Amersham Biosciences) and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories). .. Protein expression data were quantified with Quantity One Software (Bio-Rad Laboratories).

Modification:

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages
Article Snippet: Stability of siRNAs in 100% FBS 7.5 μ L of unmodified and modified siRNAs (20 μ M) were incubated with 75 μ L of FBS at 37°C. .. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA electrophoretic bands were quantified by Image Lab software (Bio-Rad).

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: EMSA was performed by using Light Chemiluminescent EMSA kit (Thermo Scientific) and following the manufacturer’s protocol with some modification. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Western Blot:

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Paragraph title: 2DE, Western blot and image analysis ... Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure.

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: Paragraph title: Electrophoresis and western blotting ... After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad).

Article Title: Therapeutic Hypothermia Activates the Endothelin and Nitric Oxide Systems after Cardiac Arrest in a Pig Model of Cardiopulmonary Resuscitation
Article Snippet: Paragraph title: Western blot ... Protein band densities were digitally quantified and normalized to the loading control (β-actin) with a ChemiDoc XRS and Quantity One image analysis software (Bio-Rad laboratories, USA).

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly
Article Snippet: Paragraph title: Western blot analysis ... After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad).

Article Title: Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue
Article Snippet: Paragraph title: Western blot ... The results were then quantified using the ChemiDoc™ XRS from BIO-RAD and the Quantity One™ software.

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: Paragraph title: SDS-PAGE and Western Blotting ... Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad).

Article Title: Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine
Article Snippet: Paragraph title: 4.8. Western Blot ... The membrane was blocked by 5% BSA and incubated with the primary antibodies on a rocker overnight, followed by incubating with the secondary antibodies on a rocker for 1 h. After rinsing four times (10 min × 1 and 5 min × 3), the membrane was developed by Millipore ECL chemiluminescence kit (Millipotre Corporation, Billerica, MA, USA) and exposure in ChemiDoc™ XRS+ Molecular Imager (Bio-Rad).

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Paragraph title: Western blot analysis ... Immune complexes formed were visualized by enhanced chemiluminescence and autoradiography (Amersham Biosciences) and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Transfection:

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: Briefly nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in 1 × binding buffer containing 5 mM MgCL2 , 25 μM ZnSO4 , 2.5% glycerol, 50 ng/ml poly(dI-dC) and proteinase inhibitor cocktail in the presence and absence of the CTCF antibody for 30 min at room temperature. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Cell Culture:

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression
Article Snippet: Briefly, bacterial strains were cultured in THY supplemented with 10% fetal bovine serum to an OD600 nm of 0.2, plated on BAP for CFU enumeration and 1 mL bacteria was pelleted and stored at −20 °C until further use. .. Membranes were developed with enhanced chemiluminiscence (ECL) detection (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA).

Generated:

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure. .. Histograms were generated using Prism V4.03 software (GraphPad Inc., San Diego, CA).

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad). .. The band volume (pixel density × area) was analyzed by Image Lab software version 4.1 (Bio-Rad), and fold changes were generated by calculating the sialidase-treated lubricin band volume divided by the untreated lubricin band volume.

Sonication:

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: Protein samples from rat hippocampal neuronal cultures were sonicated and heated at 95°C for 5 min in half the final volume of SDS-PAGE sample buffer (Tris-HCl, 62.5 mmol/L; DTT, 50 mmol/L; SDS, 2%; glycerol, 10%; and bromophenol blue, 0.1%). .. After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad).

Binding Assay:

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly
Article Snippet: Non-specific binding sites were blocked in 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) for 1 h or overnight, and the membrane was immunostained for 1 h. The antibodies used were rabbit IgG anti-LDLR (1:1000, Progen Biotechnik GmbH, Heidelberg, Germany), rabbit IgG anti-PCSK9 (1:200, Cayman Chemical Company, Ann Arbor, MI), mouse anti-human transferrin receptor (1:1000, Zymed, San Fransisco, CA), mouse IgG anti-human CD71 (1:1000, Nordic Biosite, Täby, Sweden) and mouse anti-FLAG M2 monoclonal antibody (1:3000, Sigma-Aldrich Corp., St. Louis, MO). .. After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad).

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: Briefly nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in 1 × binding buffer containing 5 mM MgCL2 , 25 μM ZnSO4 , 2.5% glycerol, 50 ng/ml poly(dI-dC) and proteinase inhibitor cocktail in the presence and absence of the CTCF antibody for 30 min at room temperature. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Molecular Weight:

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: Molecular weight standards (Precision Plus Protein Kaleidoscope Prestained Protein Standards, catalog number: 161-0375; Bio-Rad) were used on each gel. .. Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad).

Nucleic Acid Electrophoresis:

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly
Article Snippet: Cell lysates equivalent to 20 μg protein, or membrane fractions equivalent to 10 or 15 μg were separated by gel electrophoresis using a 4–20% Tris-HCl Criterion Precast Gel (Bio-Rad, Hercules, CA). .. After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad).

Labeling:

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad). .. The sequences of target DNA oligos: (1) DNA with wild type CTCF binding site: ATGCGTTTCCCCGCGAGGTGGCGCTTTCTCCCC. (2) DNA with mutated CTCF binding site: ATGCGTTTCCCCTCTCGGTGGAGCTTTCTCCCC.

Purification:

Article Title: Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue
Article Snippet: After a blocking period (milk 5% TBS tween 0.1%, 1 h), blots obtained after transfer on nitrocellulose membranes were incubated with purified home-made anti-PEPCK , anti-11β-HSD1 (Chemicon International Inc., Billerica, MA), anti-C/EBPα, anti-C/EBPβ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-C/EBPβ phosphorylated on ser105 (P-C/EBPβ) (Cell Signaling Technology, Inc., Beverly, MA) and anti-actin (Chemicon International Inc.) antibodies overnight at 4°C. .. The results were then quantified using the ChemiDoc™ XRS from BIO-RAD and the Quantity One™ software.

Dot Blot:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Equal amounts of total proteins from each tissue were used for dot blot analysis by spotting the samples through circular templates directly on to the nitrocellulose membrane. .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Polyacrylamide Gel Electrophoresis:

Article Title: Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink
Article Snippet: .. Products of were separated using 20% denaturing PAGE and detected at 526 nm using the Typhoon Imager (GE Healthcare) or ChemiDoc-XRS (BioRad). .. Pso2 has been shown to have endonuclease activity in vitro and in vivo ( , ).

Staining:

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages
Article Snippet: Samples were subjected to electrophoresis in 15% polyacrylamide-tris-borate-EDTA (TBE) under nondenaturing conditions and visualized by ethidium bromide staining. .. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA electrophoretic bands were quantified by Image Lab software (Bio-Rad).

SDS Page:

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Samples were applied on 7 cm IPG strips 3–10 NL by in-gel rehydration at 18 °C; focusing and second dimension (12% acrylamide SDS-PAGE) were performed as described . .. Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure.

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: Protein samples from rat hippocampal neuronal cultures were sonicated and heated at 95°C for 5 min in half the final volume of SDS-PAGE sample buffer (Tris-HCl, 62.5 mmol/L; DTT, 50 mmol/L; SDS, 2%; glycerol, 10%; and bromophenol blue, 0.1%). .. After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad).

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: Paragraph title: SDS-PAGE and Western Blotting ... Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad).

Article Title: Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine
Article Snippet: The electrophoresis was conducted on a SDS-PAGE gel at 120 V using Powerpac Basic electrometer (Bio-Rad, Hercules, CA, USA) following the Bio-Rad Laboratories handbook. .. The membrane was blocked by 5% BSA and incubated with the primary antibodies on a rocker overnight, followed by incubating with the secondary antibodies on a rocker for 1 h. After rinsing four times (10 min × 1 and 5 min × 3), the membrane was developed by Millipore ECL chemiluminescence kit (Millipotre Corporation, Billerica, MA, USA) and exposure in ChemiDoc™ XRS+ Molecular Imager (Bio-Rad).

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Western blot analysis Samples were run on SDS/PAGE (12%) and then electroblotted on to PVDF membranes (ImmobilonTM, Millipore). .. Immune complexes formed were visualized by enhanced chemiluminescence and autoradiography (Amersham Biosciences) and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Plasmid Preparation:

Article Title: HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
Article Snippet: Briefly nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in 1 × binding buffer containing 5 mM MgCL2 , 25 μM ZnSO4 , 2.5% glycerol, 50 ng/ml poly(dI-dC) and proteinase inhibitor cocktail in the presence and absence of the CTCF antibody for 30 min at room temperature. .. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad).

Software:

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages
Article Snippet: .. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA electrophoretic bands were quantified by Image Lab software (Bio-Rad). ..

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure. .. Histograms were generated using Prism V4.03 software (GraphPad Inc., San Diego, CA).

Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons
Article Snippet: After washing in TBST, membranes were submitted to a chemiluminescent reaction using ECL (GE Healthcare, Ecublens, Switzerland), and then photographed using the ChemiDoc XRS + (BioRad). .. Quantification was made using ImageJ software (NIH, Maryland, USA).

Article Title: Therapeutic Hypothermia Activates the Endothelin and Nitric Oxide Systems after Cardiac Arrest in a Pig Model of Cardiopulmonary Resuscitation
Article Snippet: .. Protein band densities were digitally quantified and normalized to the loading control (β-actin) with a ChemiDoc XRS and Quantity One image analysis software (Bio-Rad laboratories, USA). .. Immunohistochemistry (IHC) Fixation: Cardiac left ventricle tissue was immersed in 4% buffered formalin and stored one week at 4°C before small tissue pieces ( < 3×5 mm) from the were cut and processed for immunohistochemistry.

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly
Article Snippet: After two more washing steps with TBS containing 0.1% Tween-20, the bands were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad). .. Quantity One version 4.4.0 software (Bio-Rad) was used for quantification of band intensities.

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression
Article Snippet: .. Membranes were developed with enhanced chemiluminiscence (ECL) detection (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA). .. Proteomics Total proteins were isolated from mid-log phase (OD600 nm 0.4) TIGR4, and ΔcadA (n = 4) cultured in THY and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis as described earlier [ , ].

Article Title: Central Glucocorticoid Administration Promotes Weight Gain and Increased 11?-Hydroxysteroid Dehydrogenase Type 1 Expression in White Adipose Tissue
Article Snippet: .. The results were then quantified using the ChemiDoc™ XRS from BIO-RAD and the Quantity One™ software. ..

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories). .. Chemiluminescence was quantified using Quantity One Software (Bio-Rad Laboratories) and the acquisition data were performed under conditions to prevent saturation of the chemiluminescence signal.

Article Title: Sialidase Unmasks Mucin Domain Epitopes of Lubricin
Article Snippet: Membranes were incubated with secondary antibody (goat antimouse IgG, 1:10,000, catalog number: 31432; Thermo Fisher Scientific) in PBS and 0.05% Tween-20, pH 7.4, for 1 hr at room temperature, developed with SuperSignal West Pico chemiluminescent substrate (catalog number: 34077; Thermo Fisher Scientific), and imaged (ChemiDoc XRS+; Bio-Rad). .. The band volume (pixel density × area) was analyzed by Image Lab software version 4.1 (Bio-Rad), and fold changes were generated by calculating the sialidase-treated lubricin band volume divided by the untreated lubricin band volume.

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Immune complexes formed were visualized by enhanced chemiluminescence and autoradiography (Amersham Biosciences) and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories). .. Protein expression data were quantified with Quantity One Software (Bio-Rad Laboratories).

Homogenization:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Tissue samples were homogenized in four volumes of ice-cold homogenization buffer (10 mM Tris/HCl, pH 7.5, containing 150 mM NaCl) with an Ultra-Turrax T25 homogenizer (IKAWorks). .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Produced:

Article Title: Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii
Article Snippet: Following blocking, the membrane was incubated with Ub conjugates specific primary antibody HRP conjugate (1:2500, produced by Enzo Life Sciences) for 1 h at room temperature. .. Enhanced chemiluminescence and autoradiography (Amersham Biosciences) were used to displayed immune complexes formed and measured by densitometry analysis with ChemiDoc XRS (Bio-Rad Laboratories).

Concentration Assay:

Article Title: The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages
Article Snippet: J774A.1 cells (1×106 ) were exposed to 50 μM HOSCN for 15 min, then cell proteins were extracted and the concentration determined (Lowry assay, Bio-Rad). .. Signals were directly digitized using a ChemiDoc XRS (Bio-Rad).

Two-Dimensional Gel Electrophoresis:

Article Title: A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Article Snippet: Paragraph title: 2DE, Western blot and image analysis ... Blot images were acquired using ChemiDoc XRS + (BioRad) or using film exposure.

Lysis:

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression
Article Snippet: CPS was extracted in a lysis buffer (4% deoxycholate, 50 μg/mL DNAseI and 50 μg/mL RNAse) at 37 °C for 10 min and centrifuged at 18,000× g for 10 min. Four threefold serial dilutions of the supernatant in phosphate-buffered saline (PBS) were spotted in duplicate (2 μL) on 0.2-μm-pore-size nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA, USA) with suction and air dried at 60 °C for 15 min. .. Membranes were developed with enhanced chemiluminiscence (ECL) detection (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA).

Variant Assay:

Article Title: The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression
Article Snippet: An isogenic capsular variant of TIGR4 (T4R) in which the cps locus is replaced with the Janus cassette resulting in an unencapsulated phenotype [ ] was used as a control. .. Membranes were developed with enhanced chemiluminiscence (ECL) detection (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA).

Ab Array:

Article Title: The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages
Article Snippet: Paragraph title: Phospho-MAPK antibody array ... Signals were directly digitized using a ChemiDoc XRS (Bio-Rad).

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    Bio-Rad chemidoc xrs system
    Selective labeling of sfGFPs that contained site-specifically incorporated NCAAs with strained alkene/alkyne functionalities with (A) FITC-TZ and (B) HZCL. In A and B, the top panels show denaturing SDS-PAGE analysis of proteins stained with Coomassie blue and the bottom panels are from fluorescent imaging of the same gels before Coomassie blue staining. In A, the fluorescent image was captured by a digital camera and displayed was real color of the emitting light. In B, the fluorescent image was captured by a Bio-Rad <t>ChemiDoc</t> <t>XRS</t> system.
    Chemidoc Xrs System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selective labeling of sfGFPs that contained site-specifically incorporated NCAAs with strained alkene/alkyne functionalities with (A) FITC-TZ and (B) HZCL. In A and B, the top panels show denaturing SDS-PAGE analysis of proteins stained with Coomassie blue and the bottom panels are from fluorescent imaging of the same gels before Coomassie blue staining. In A, the fluorescent image was captured by a digital camera and displayed was real color of the emitting light. In B, the fluorescent image was captured by a Bio-Rad ChemiDoc XRS system.

    Journal: Bioconjugate Chemistry

    Article Title: Two Rapid Catalyst-Free Click Reactions for In Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities

    doi: 10.1021/bc500361d

    Figure Lengend Snippet: Selective labeling of sfGFPs that contained site-specifically incorporated NCAAs with strained alkene/alkyne functionalities with (A) FITC-TZ and (B) HZCL. In A and B, the top panels show denaturing SDS-PAGE analysis of proteins stained with Coomassie blue and the bottom panels are from fluorescent imaging of the same gels before Coomassie blue staining. In A, the fluorescent image was captured by a digital camera and displayed was real color of the emitting light. In B, the fluorescent image was captured by a Bio-Rad ChemiDoc XRS system.

    Article Snippet: The fluorescence was detected in a Bio-Rad ChemiDoc XRS+ system under UV irradiation.

    Techniques: Labeling, SDS Page, Staining, Imaging

    Pseudotyped particle protein density analysis. Quantification of intensities of HA, NA and VSV M protein by Western Blot ( A ). The HA 1 ( B ) and NA ( C ) band intensities were directly detected by Chemidoc XRS+ imager and normalized to corresponding M band intensity to obtain the relative HA and NA expression levels on different VSV pseudotyped particles. Each error bar represents the mean ± SD of three independent experiments. NA protein was also detected except for HA only particles.

    Journal: Scientific Reports

    Article Title: Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

    doi: 10.1038/srep35537

    Figure Lengend Snippet: Pseudotyped particle protein density analysis. Quantification of intensities of HA, NA and VSV M protein by Western Blot ( A ). The HA 1 ( B ) and NA ( C ) band intensities were directly detected by Chemidoc XRS+ imager and normalized to corresponding M band intensity to obtain the relative HA and NA expression levels on different VSV pseudotyped particles. Each error bar represents the mean ± SD of three independent experiments. NA protein was also detected except for HA only particles.

    Article Snippet: All Western blots were visualized and analyzed using a Chemidoc XRS+ system with Image Lab image capture software (BioRad).

    Techniques: Western Blot, Expressing

    Construction and verification of a qcrABC deletion mutant. ( A ) Mutagenesis strategy. The majority of the coding regions of the qcrA-C genes were replaced with a kanamycin resistance cassette with its own promoter. The downstream cfa gene has its own promoter, but the cassette has no terminator and so should be non-polar on cfa . ( B ) RT-PCR to verify expression of cfa gene and absence of qcr gene transcription in the qcrABC mutant. Fold-change in the mutant is shown relative to the wild type strain, using gyrA gene as a control. The difference between expression of the cfa gene in mutant and wild-type was not significant by t-test. ( C ) shows a comparison of the membrane associated c -type cytochromes revealed after a total membrane preparation was subjected to SDS-PAGE and electroblotting to a nitrocellulose membrane, followed by staining for haem-associated peroxidase activity using the chemiluminescence technique described in Methods. 20 μg total protein was run per lane. The Image shown is a cropped version of the full-size blot that can viewed in Supplementary Fig. 1 , and was obtained using a ChemiDoc XRS system (BioRad Inc) with an exposure time of 2 min. A band corresponding to the expected size of QcrC is missing in the qcrABC deletion mutant.

    Journal: Scientific Reports

    Article Title: Bacterial periplasmic nitrate and trimethylamine-N-oxide respiration coupled to menaquinol-cytochrome c reductase (Qcr): Implications for electrogenic reduction of alternative electron acceptors

    doi: 10.1038/s41598-018-33857-2

    Figure Lengend Snippet: Construction and verification of a qcrABC deletion mutant. ( A ) Mutagenesis strategy. The majority of the coding regions of the qcrA-C genes were replaced with a kanamycin resistance cassette with its own promoter. The downstream cfa gene has its own promoter, but the cassette has no terminator and so should be non-polar on cfa . ( B ) RT-PCR to verify expression of cfa gene and absence of qcr gene transcription in the qcrABC mutant. Fold-change in the mutant is shown relative to the wild type strain, using gyrA gene as a control. The difference between expression of the cfa gene in mutant and wild-type was not significant by t-test. ( C ) shows a comparison of the membrane associated c -type cytochromes revealed after a total membrane preparation was subjected to SDS-PAGE and electroblotting to a nitrocellulose membrane, followed by staining for haem-associated peroxidase activity using the chemiluminescence technique described in Methods. 20 μg total protein was run per lane. The Image shown is a cropped version of the full-size blot that can viewed in Supplementary Fig. 1 , and was obtained using a ChemiDoc XRS system (BioRad Inc) with an exposure time of 2 min. A band corresponding to the expected size of QcrC is missing in the qcrABC deletion mutant.

    Article Snippet: Images were obtained using a ChemiDoc XRS system (BioRad Inc) with an exposure time of 2 min.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, SDS Page, Staining, Activity Assay

    Western blot analysis of extracellular matrix components in wound exudates collected from mice after injection of B . asper venom. Groups of five mice were injected in the gastrocnemius with 50 μg of B . asper venom. After 1, 6 and 24 h of injection mice were sacrificed and samples of exudates were collected, pooled and lyophilized. Afterwards, 100 μg of protein of each sample were separated under reducing conditions on 4–15% Tris–HCl SDS-PAGE, and transferred to nitrocellulose membranes. Immunodetection was performed with (A) anti-type IV collagen (Col IV), (B) anti-laminin, (C) anti-nidogen 1, (D) anti-type I collagen (Col I), (E) anti-type VI collagen (Col VI), and (F) anti-fibronectin. The reaction was detected using an anti-rabbit peroxidase antibody and a chemiluminescent substrate. Images were obtained with the ChemiDoc XRS+ System (BioRad).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

    doi: 10.1371/journal.pntd.0004599

    Figure Lengend Snippet: Western blot analysis of extracellular matrix components in wound exudates collected from mice after injection of B . asper venom. Groups of five mice were injected in the gastrocnemius with 50 μg of B . asper venom. After 1, 6 and 24 h of injection mice were sacrificed and samples of exudates were collected, pooled and lyophilized. Afterwards, 100 μg of protein of each sample were separated under reducing conditions on 4–15% Tris–HCl SDS-PAGE, and transferred to nitrocellulose membranes. Immunodetection was performed with (A) anti-type IV collagen (Col IV), (B) anti-laminin, (C) anti-nidogen 1, (D) anti-type I collagen (Col I), (E) anti-type VI collagen (Col VI), and (F) anti-fibronectin. The reaction was detected using an anti-rabbit peroxidase antibody and a chemiluminescent substrate. Images were obtained with the ChemiDoc XRS+ System (BioRad).

    Article Snippet: Images were captured with the ChemiDoc XRS+ System (BioRad) and the analysis was performed with the ImageLab software (BioRad).

    Techniques: Western Blot, Mouse Assay, Injection, SDS Page, Immunodetection