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Bio-Rad tris hcl
Reaction of the reduced WT ThyX·5-dUMPS complex with CH 2 THF. Anaerobic solutions of reduced WT ThyX (14 μM active sites) and dUMP [300 μM (black)] or with 5-dUMPS [300 μM (blue)] were mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer. The reaction mixtures were in 0.1 M <t>Tris-HCl</t> (pH 8.0) with 1 mM <t>EDTA</t> and 15 mM CH 2 O at 25 °C.

Reaction of the reduced WT ThyX·5-dUMPS complex with CH 2 THF. Anaerobic solutions of reduced WT ThyX (14 μM active sites) and dUMP [300 μM (black)] or with 5-dUMPS [300 μM (blue)] were mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer. The reaction mixtures were in 0.1 M <t>Tris-HCl</t> (pH 8.0) with 1 mM <t>EDTA</t> and 15 mM CH 2 O at 25 °C.

Tris Hcl, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation"

Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation

Journal: Biochemistry

doi: 10.1021/bi500648n

Figure Legend Snippet: Reaction of the reduced WT ThyX·5-dUMPS complex with CH 2 THF. Anaerobic solutions of reduced WT ThyX (14 μM active sites) and dUMP [300 μM (black)] or with 5-dUMPS [300 μM (blue)] were mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C.

Techniques Used: Flow Cytometry, Spectrophotometry

Spectrum of the intermediate detected in the oxidative half-reaction. An anaerobic solution of reduced WT ThyX (14 μM active sites, after mixing) and dUMP (300 μM) in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA was mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer in diode-array mode. (A) Stereoview of spectra as a function of time. Spectra were recorded with an integration time of 1.5 ms at various intervals out to 10 s. Note the logarithmic time scale. (B) Deconvoluted intermediate spectra calculated by singular-value decomposition using the raw data in panel A and a two-step mechanism, giving rate constants of 28.4 and 0.2 s –1 .

Figure Legend Snippet: Spectrum of the intermediate detected in the oxidative half-reaction. An anaerobic solution of reduced WT ThyX (14 μM active sites, after mixing) and dUMP (300 μM) in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA was mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer in diode-array mode. (A) Stereoview of spectra as a function of time. Spectra were recorded with an integration time of 1.5 ms at various intervals out to 10 s. Note the logarithmic time scale. (B) Deconvoluted intermediate spectra calculated by singular-value decomposition using the raw data in panel A and a two-step mechanism, giving rate constants of 28.4 and 0.2 s –1 .

Techniques Used: Flow Cytometry, Spectrophotometry, Mass Spectrometry

Chemical quenching. (A) An anaerobic solution of reduced WT ThyX (50 μM) and dUMP (300 μM) was mixed with 400 μM CH 2 THF and 15 mM formaldehyde in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA. The reaction was quenched with 1 M HCl at different times, and the concentrations of dUMP (green) and dTMP (orange) were calculated from the area under the peaks of HPLC chromatograms. An absorbance trace at 420 nm obtained in stopped-flow experiments (black) is shown for comparison. The vertical lines at 0.13 and 2.6 s indicate the times of maximal accumulation of intermediates I 1 , detected by the flavin spectral change, and I 2 , detected by the consumption of dUMP. (B) Calculated concentrations of species during the oxidative half-reaction. The rate constants from stopped-flow experiments, 28.4 and 0.2 s –1 , and the rate constant from the consumption of dUMP, 0.7 s –1 , observed by quenching, were used to simulate consecutive reactions. The simulation used an enzyme concentration of 20 μM and shows that intermediates accumulate maximally at 0.13 and 2.6 s.

Figure Legend Snippet: Chemical quenching. (A) An anaerobic solution of reduced WT ThyX (50 μM) and dUMP (300 μM) was mixed with 400 μM CH 2 THF and 15 mM formaldehyde in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA. The reaction was quenched with 1 M HCl at different times, and the concentrations of dUMP (green) and dTMP (orange) were calculated from the area under the peaks of HPLC chromatograms. An absorbance trace at 420 nm obtained in stopped-flow experiments (black) is shown for comparison. The vertical lines at 0.13 and 2.6 s indicate the times of maximal accumulation of intermediates I 1 , detected by the flavin spectral change, and I 2 , detected by the consumption of dUMP. (B) Calculated concentrations of species during the oxidative half-reaction. The rate constants from stopped-flow experiments, 28.4 and 0.2 s –1 , and the rate constant from the consumption of dUMP, 0.7 s –1 , observed by quenching, were used to simulate consecutive reactions. The simulation used an enzyme concentration of 20 μM and shows that intermediates accumulate maximally at 0.13 and 2.6 s.

Techniques Used: High Performance Liquid Chromatography, Flow Cytometry, Concentration Assay

Oxidative half-reactions of variant enzymes. The reduced variant enzyme·dUMP complexes were mixed with saturating concentrations CH 2 THF using a stopped-flow spectrophotometer. The reactions were monitored by their absorbance at 420 nm. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C. Traces, labeled by ThyX variant, are displayed in two panels for the sake of clarity.

Figure Legend Snippet: Oxidative half-reactions of variant enzymes. The reduced variant enzyme·dUMP complexes were mixed with saturating concentrations CH 2 THF using a stopped-flow spectrophotometer. The reactions were monitored by their absorbance at 420 nm. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C. Traces, labeled by ThyX variant, are displayed in two panels for the sake of clarity.

Techniques Used: Variant Assay, Flow Cytometry, Spectrophotometry, Labeling

2) Product Images from "Evidence for Convergent Evolution in the Signaling Properties of a Choanoflagellate Tyrosine Kinase †"

Article Title: Evidence for Convergent Evolution in the Signaling Properties of a Choanoflagellate Tyrosine Kinase †

Journal:

doi: 10.1021/bi9000672

Effect of autophosphorylation on MbSrc4 activity. MbSrc4 (5 μ L of 68 μ M) was incubated with immobilized GST-YOP in 50 mM Tris (pH 7.5) and 50 mM NaCl (200 μ L final volume). The reaction was mixed at room temperature for 30 min.

Figure Legend Snippet: Effect of autophosphorylation on MbSrc4 activity. MbSrc4 (5 μ L of 68 μ M) was incubated with immobilized GST-YOP in 50 mM Tris (pH 7.5) and 50 mM NaCl (200 μ L final volume). The reaction was mixed at room temperature for 30 min.

Techniques Used: Activity Assay, Incubation

SDS Page:

Article Title: Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo
Article Snippet: .. Alternatively, reactions were terminated by addition of SDS-sample buffer followed by SDS/PAGE using 18% Tris⋅HCl (acrylamide:bisacrylamide 120:1) gels or precast 16.5% Tris-Tricine peptide gels (Bio-Rad). .. After staining of the histones with Coomassie blue, the gels were subjected to fluorography.

Article Title: Decreased function of survival motor neuron protein impairs endocytic pathways
Article Snippet: Cells were centrifuged at 855 × g for 5 min and pellets were resuspended in 1× RIPA buffer with protease (1:10) and phosphatase (1:100) inhibitors (Sigma) and incubated on ice for 30 min. .. Cells were pelleted at 18,600 × g , and supernatants were diluted at a 1:1 ratio in SDS loading buffer, boiled at 95 °C for 5 min, and 15 µL was resolved by SDS/PAGE using a 4–15% (vol/vol) Tris·HCl gel (BioRad). .. Gels were transferred to PVDF membranes (BioRad) using a Transblot system (BioRad) at 10 V for 30 min. Membranes were blocked in 2% (vol/vol) milk in PBS with 0.1% Tween 20% (vol/vol) (PBS-T) O/N, then incubated with a purified mouse anti-SMN primary antibody at 1:5,000 dilution (BD Biosciences; 610647) and an anti-alpha tubulin polyclonal antibody loading control at 1:450 dilution (Abcam; ab4074) at RT for 1 h, washed in PBS-T, and then incubated with a 680 nM goat–anti-mouse secondary antibody at 1:1,000 dilution (Life Technologies) and a 800 nM anti-rabbit secondary antibody at 1:5,000 dilution (LI-COR Biosciences) at RT for 1 h. All antibodies were diluted in 2% (vol/vol) milk.

Article Title: Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport
Article Snippet: Membrane unstacking involved incubation of the intact thylakoid preparation in Mg2+ -free WB containing 50 mM EDTA for 15 min, followed by two times wash with Mg2+ -free/EDTA-free WB solution and the tryptic digestion in Mg2+ -free reaction mixture. .. Protein samples for SDS/PAGE were incubated in 50 mM Tris⋅HCl (pH 6.8) containing 2% (wt/vol) SDS, 6 M urea, 50 mM DTT, 1 mM PMSF, 5 mM EDTA, 10% (wt/vol) glycerol, and 0.05% (wt/vol) bromophenol blue at 95 °C for 3 min and separated on 4–20% Mini-PROTEAN TGX Stain-Free SDS/PAGE (Bio-Rad) in Mini-PROTEAN Tetra Cell (Bio-Rad). .. For immunoblot analyses, proteins were transferred to polyvinylidene fluoride (PVDF) membrane by semidry electroblotting in Trans-Blot Turbo Transfer System (BioRad).

Polymerase Chain Reaction:

Article Title: Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules
Article Snippet: The equivalent of 0.83 μL of the cDNA generated in its synthesis step was used per reaction. .. PCR reactions were performed in the final volume of 25 μL containing 20 mM Tris⋅HCl (pH 8.4), 50 mM KCl, 200 μM of each of four dNTPs (dATP, dCTP, dGTP, dTTP), 0.5 μM of each primer, 3.5 mM MgCl2 , and 1 × SYBR Green (Bio-Rad). .. Six samples for each treatment group and PCR assays in duplicate were used.

SYBR Green Assay:

Protein Concentration:

Article Title: Immunoglobulin G from bovine milk primes intestinal epithelial cells for increased colonization of bifidobacteria
Article Snippet: The column was washed with 5 column volumes (25 mL) of binding buffer and IgG was eluted with elution buffer (100 mM glycine–HCl, pH 2.7). .. The recovered IgG-containing fractions were immediately neutralized with 1 M Tris⁄HCl (pH 9) and the protein concentration was determined using the Quick Start Bovine γ-Globulin Standard (BioRad Laboratories Ltd.). .. Fractions with the highest concentration of IgG from each run were pooled.

Article Title: Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia
Article Snippet: Western blotting was used to determine which receptors for VEGF were expressed by HN33 cells in culture. .. Cell lysates were extracted in 0.1 M NaCl, 0.01 M Tris⋅HCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 μg/ml aprotinin, and 100 μg/ml PMSF , and protein concentration was determined by a Bio-Rad protein assay. .. Protein (100 μg) was boiled at 100°C in SDS sample buffer for 5 min, electrophoresed on 7–12% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes.

Incubation:

Staining:

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acrylamide tris hcl ready sds page gels (Bio-Rad)

Bio-Rad acrylamide tris hcl ready sds page gels

Figure 1. Effect of LAs on temperature dependence of KcsA tetramer stability in the presence and absence of K + . KcsA tetramer was measured by <t>SDS-PAGE</t> analysis of a series of identical samples incubated at various temperatures for 10 min before addition of SDS-PAGE sample buffer. The sample assay mixture contained 10 mM <t>Hepes-Tris,</t> pH 7.4, 100 mM cholineCl, either 5 mM KCl ( A ) or no added KCl ( B ) and either no LA (○), 20 mM lidocaine (●) or 5 mM tetracaine (△). Solid lines indicate nonlinear regression fits to a logistic function of temperature described in Materials and Methods . Fit parameters: ( A ) 20 mM lidocaine (●) : T 0.5 = 98.0 ± 2.1 °C, n = 14.1 ± 4.8; ( A ) 5 mM tetracaine (△): T 0.5 = 75.9 ± 1.7 °C, n = 10.7 ± 2.3; ( B ) no LA (○): T 0.5 = 45.7 ± 0.9 °C, n = 6.4 ± 0.7; ( B ) 20 mM lidocaine (●) : T 0.5 = 41.9 ± 0.7 °C, n = 6.4 ± 0.6.

Acrylamide Tris Hcl Ready Sds Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 1. Effect of LAs on temperature dependence of KcsA tetramer stability in the presence and absence of K + . KcsA tetramer was measured by SDS-PAGE analysis of a series of identical samples incubated at various temperatures for 10 min before addition of SDS-PAGE sample buffer. The sample assay mixture contained 10 mM Hepes-Tris, pH 7.4, 100 mM cholineCl, either 5 mM KCl ( A ) or no added KCl ( B ) and either no LA (○), 20 mM lidocaine (●) or 5 mM tetracaine (△). Solid lines indicate nonlinear regression fits to a logistic function of temperature described in Materials and Methods . Fit parameters: ( A ) 20 mM lidocaine (●) : T 0.5 = 98.0 ± 2.1 °C, n = 14.1 ± 4.8; ( A ) 5 mM tetracaine (△): T 0.5 = 75.9 ± 1.7 °C, n = 10.7 ± 2.3; ( B ) no LA (○): T 0.5 = 45.7 ± 0.9 °C, n = 6.4 ± 0.7; ( B ) 20 mM lidocaine (●) : T 0.5 = 41.9 ± 0.7 °C, n = 6.4 ± 0.6.

Journal: Channels

Article Title: Interaction of local anesthetics with the K+ channel pore domain

doi: 10.4161/chan.24455

Figure Lengend Snippet: Figure 1. Effect of LAs on temperature dependence of KcsA tetramer stability in the presence and absence of K + . KcsA tetramer was measured by SDS-PAGE analysis of a series of identical samples incubated at various temperatures for 10 min before addition of SDS-PAGE sample buffer. The sample assay mixture contained 10 mM Hepes-Tris, pH 7.4, 100 mM cholineCl, either 5 mM KCl ( A ) or no added KCl ( B ) and either no LA (○), 20 mM lidocaine (●) or 5 mM tetracaine (△). Solid lines indicate nonlinear regression fits to a logistic function of temperature described in Materials and Methods . Fit parameters: ( A ) 20 mM lidocaine (●) : T 0.5 = 98.0 ± 2.1 °C, n = 14.1 ± 4.8; ( A ) 5 mM tetracaine (△): T 0.5 = 75.9 ± 1.7 °C, n = 10.7 ± 2.3; ( B ) no LA (○): T 0.5 = 45.7 ± 0.9 °C, n = 6.4 ± 0.7; ( B ) 20 mM lidocaine (●) : T 0.5 = 41.9 ± 0.7 °C, n = 6.4 ± 0.6.

Article Snippet: Ten milliliters of each sample was pipetted into wells of precast 12% acrylamide TRIS-HCl ready SDS-PAGE Gels (Bio-Rad) and subjected to electrophoresis at 200V for ~35 min in running buffer (25 mM Tris base, 192 mM glycine, pH 8.3, 0.1% SDS).

Techniques: SDS Page, Incubation

Figure 2. Destabilization of KcsA tetramer as function of LA concentration. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing concentrations of lidocaine (●) or tetracaine (△, □ ). The sample assay mixture contained 10 mM Hepes-Tris, pH 7.5, 100 mM cholineCl and either 5 mM KCl (● , △) or no added KCl (□ ). Samples were incubated either at 90°C (● , △) or 22 °C (□ ) for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of LA concentration as described in Materials and Methods . Fit parameters: lidocaine, 5 mM KCl, 90°C (● ): IC 50 = 25.1 ± 2.6 mM, n = 1.14 ± 0.13; tetracaine, 5 mM KCl, 90 °C (△): IC 50 = 4.2 ± 0.6 mM, n = 1.43 ± 0.33; tetracaine, 0 KCl, 22 °C (□ ): 1.2 ± 0.2 mM, n = 0.98 ± 0.22.

Journal: Channels

Article Title: Interaction of local anesthetics with the K+ channel pore domain

doi: 10.4161/chan.24455

Figure Lengend Snippet: Figure 2. Destabilization of KcsA tetramer as function of LA concentration. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing concentrations of lidocaine (●) or tetracaine (△, □ ). The sample assay mixture contained 10 mM Hepes-Tris, pH 7.5, 100 mM cholineCl and either 5 mM KCl (● , △) or no added KCl (□ ). Samples were incubated either at 90°C (● , △) or 22 °C (□ ) for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of LA concentration as described in Materials and Methods . Fit parameters: lidocaine, 5 mM KCl, 90°C (● ): IC 50 = 25.1 ± 2.6 mM, n = 1.14 ± 0.13; tetracaine, 5 mM KCl, 90 °C (△): IC 50 = 4.2 ± 0.6 mM, n = 1.43 ± 0.33; tetracaine, 0 KCl, 22 °C (□ ): 1.2 ± 0.2 mM, n = 0.98 ± 0.22.

Techniques: Concentration Assay, SDS Page, Incubation

Figure 3. Stabilization of KcsA tetramer as a function of K + concentration in the absence and presence of LA. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing K + concentration in the absence of LA (○) or in the presence of 20 mM lidocaine (● ) or 5 mM tetracaine (△). The sample assay mixture contained ~500 ng KcsA, 10 mM Hepes-Tris, pH 7.4, 100 mM choline Cl, indicated concentrations of KCl and either no LA, 20 mM lidocaine or 5 mM tetracaine. All samples were incubated at 90 °C for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of K + concentration as described in Materials and Methods . Fit parameters: no LA (○): K 0.5 = 1.48 ± 0.04 mM, n = 3.19 ± 0.26; 20 mM lidocaine (● ): K 0.5 = 6.65 ± 0.8 mM, n = 1.05 ± 0.13; 5 mM tetracaine (△): K 0.5 = 5.03 ± 0.90, n = 1.04 ± 0.19.

Journal: Channels

Article Title: Interaction of local anesthetics with the K+ channel pore domain

doi: 10.4161/chan.24455

Figure Lengend Snippet: Figure 3. Stabilization of KcsA tetramer as a function of K + concentration in the absence and presence of LA. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing K + concentration in the absence of LA (○) or in the presence of 20 mM lidocaine (● ) or 5 mM tetracaine (△). The sample assay mixture contained ~500 ng KcsA, 10 mM Hepes-Tris, pH 7.4, 100 mM choline Cl, indicated concentrations of KCl and either no LA, 20 mM lidocaine or 5 mM tetracaine. All samples were incubated at 90 °C for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of K + concentration as described in Materials and Methods . Fit parameters: no LA (○): K 0.5 = 1.48 ± 0.04 mM, n = 3.19 ± 0.26; 20 mM lidocaine (● ): K 0.5 = 6.65 ± 0.8 mM, n = 1.05 ± 0.13; 5 mM tetracaine (△): K 0.5 = 5.03 ± 0.90, n = 1.04 ± 0.19.

Techniques: Concentration Assay, SDS Page, Incubation

Cystatin A inhibits the processing of MYOC wild-type in cultured cells. Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in Methods . pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.

Journal: PLoS ONE

Article Title: Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

doi: 10.1371/journal.pone.0036301

Figure Lengend Snippet: Cystatin A inhibits the processing of MYOC wild-type in cultured cells. Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in Methods . pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.

Article Snippet: Aliquots of 4 µl from either the cell lysates or the media were mixed (1∶2 vol) with Laemmli buffer (Bio-Rad, Hercules, CA) containing 5% β-mercaptoethanol and loaded onto 4–15% SDS-PAGE Tris-HCl polyacrylamide gels (Bio-Rad).

Techniques: Cell Culture, Recombinant, Expressing, Generated, Transfection, SDS Page, Western Blot, Electroporation

$Subcellular localization of DmLRPPRC2. (A) Fluorescence confocal microscopy images showing HeLa cells ( n = 15) expressing a DmLRPPRC2-GFP fusion protein and counterstained with the mitochondrial marker MitoTracker Deep Red. Scale bar: 10 μm. (B) Subcellular fractionation showing the presence of DmLRPPRC2 in the mitochondrial fraction. Subcellular fractions were separated by SDS-PAGE followed by western blot analysis to detect histone H3 (a nuclear marker), tubulin (a cytosolic marker), VDAC (a mitochondrial marker) and DmLRPPRC2. See also Supplementary Figure S1A and B.$

Journal: Nucleic Acids Research

Article Title: Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation

doi: 10.1093/nar/gku1132

Figure Lengend Snippet: Subcellular localization of DmLRPPRC2. (A) Fluorescence confocal microscopy images showing HeLa cells ( n = 15) expressing a DmLRPPRC2-GFP fusion protein and counterstained with the mitochondrial marker MitoTracker Deep Red. Scale bar: 10 μm. (B) Subcellular fractionation showing the presence of DmLRPPRC2 in the mitochondrial fraction. Subcellular fractions were separated by SDS-PAGE followed by western blot analysis to detect histone H3 (a nuclear marker), tubulin (a cytosolic marker), VDAC (a mitochondrial marker) and DmLRPPRC2. See also Supplementary Figure S1A and B.

Article Snippet: All isolated fractions were separated in 4–15% Criterion Tris-HCl precast SDS-PAGE gels (Bio-Rad) and tested by a standard western blotting procedure.

Techniques: Fluorescence, Confocal Microscopy, Expressing, Marker, Fractionation, SDS Page, Western Blot

Steady-state levels and activities of the OXPHOS complexes. (A) Western blot analysis of the Lrpprc2 RNAi#1 line (left side) and the Lrpprc2 RNAi#2 line (right side) performed on 20–40 μg whole-body protein extracts from controls and DmLrpprc2 KD third-instar larvae and 6-day-old adults. Protein extracts were separated by standard SDS-PAGE followed by western blot analysis with antibodies against the nuclear-encoded subunit NDUFS3 of complex I, the α-subunit of complex V and VDAC, the latter used as reference for loading. (B) BN-PAGE combined with complex I, complex IV and complex V in-gel activity analyses on the Lrpprc2 RNAi#1 line and the Lrpprc2 RNAi#2 line. BN-PAGE was performed on 75 μg for complex I, 100 μg for complex IV and 150 μg for complex V of mitochondrial protein extracts from control and DmLrpprc2 KD third-instar larvae. The assembly status of complex V (right panel) is black because of color inversion. Loading controls are provided by Coomassie staining to assess the total mitochondrial protein content per sample (only for complex V, left panel) and western blot analysis of VDAC protein levels in mitochondrial protein lysates collected prior to BN-PAGE gel loading. The position of complex I (CI), complex IV (CIV), complex V dimers (CV 2 ), complex V monomers (CV 1 ) and complex V subassembled components ( sub CV a and sub CV b ) are indicated by arrows.

Journal: Nucleic Acids Research

Article Title: Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation

doi: 10.1093/nar/gku1132

Figure Lengend Snippet: Steady-state levels and activities of the OXPHOS complexes. (A) Western blot analysis of the Lrpprc2 RNAi#1 line (left side) and the Lrpprc2 RNAi#2 line (right side) performed on 20–40 μg whole-body protein extracts from controls and DmLrpprc2 KD third-instar larvae and 6-day-old adults. Protein extracts were separated by standard SDS-PAGE followed by western blot analysis with antibodies against the nuclear-encoded subunit NDUFS3 of complex I, the α-subunit of complex V and VDAC, the latter used as reference for loading. (B) BN-PAGE combined with complex I, complex IV and complex V in-gel activity analyses on the Lrpprc2 RNAi#1 line and the Lrpprc2 RNAi#2 line. BN-PAGE was performed on 75 μg for complex I, 100 μg for complex IV and 150 μg for complex V of mitochondrial protein extracts from control and DmLrpprc2 KD third-instar larvae. The assembly status of complex V (right panel) is black because of color inversion. Loading controls are provided by Coomassie staining to assess the total mitochondrial protein content per sample (only for complex V, left panel) and western blot analysis of VDAC protein levels in mitochondrial protein lysates collected prior to BN-PAGE gel loading. The position of complex I (CI), complex IV (CIV), complex V dimers (CV 2 ), complex V monomers (CV 1 ) and complex V subassembled components ( sub CV a and sub CV b ) are indicated by arrows.

Article Snippet: All isolated fractions were separated in 4–15% Criterion Tris-HCl precast SDS-PAGE gels (Bio-Rad) and tested by a standard western blotting procedure.

Techniques: Western Blot, SDS Page, Polyacrylamide Gel Electrophoresis, Activity Assay, Staining

Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

Journal: Molecular and Cellular Biology

Article Title: Conditional Disruption of the Peroxisome Proliferator-Activated Receptor ? Gene in Mice Results in Lowered Expression of ABCA1, ABCG1, and apoE in Macrophages and Reduced Cholesterol Efflux

doi: 10.1128/MCB.22.8.2607-2619.2002

Figure Lengend Snippet: Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

Article Snippet: Then, 10 μg of total protein from Hepa-1 cells and 10 μg of protein from nuclear extracts of macrophages were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad, Hercules, Calif.), transferred to Immobilon-P membranes (Millipore, Bedford, Mass.), and probed according to the manufacturer's recommendations with anti-PPARγ antibodies (E-8 and H-100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) as indicated.

Techniques: Western Blot, Mouse Assay, Transfection, Expressing, Plasmid Preparation, Electrophoresis

Structured Review

Images

1) Product Images from "Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation"

2) Product Images from "Evidence for Convergent Evolution in the Signaling Properties of a Choanoflagellate Tyrosine Kinase †"

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SYBR Green Assay:

Protein Concentration:

Incubation:

Staining:

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