Structured Review

Bio-Rad tris hcl gel
Bmf S and Bmf CUG show the same interaction pattern with anti-apoptotic Bcl-2 family members (A) Bmf S and Bmf CUG were overexpressed simultaneously in 293T cells together with FLAG-tagged versions of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, BHRF1 or KS-Bcl2. Expression of transgene-derived protein was confirmed by immunoblotting using anti-FLAG- or anti-Bmf-specific mAbs (# = marker labeling). (B) Immunoprecipitation was performed using anti-FLAG-M2 mAb. Immune complexes were separated by <t>SDS-PAGE</t> on 4-20% <t>Tris-glycine</t> gels. After electroblotting, nitrocellulose membranes were subjected to immunoblotting using the anti-Bmf mAb (17A9). Membranes were stripped and reprobed with a rat-anti-FLAG antibody. One out of three independent experiments yielding similar results is shown. (* short exposure, ** long exposure)
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1) Product Images from "BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis"

Article Title: BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis

Journal: Cell death and differentiation

doi: 10.1038/cdd.2010.97

Bmf S and Bmf CUG show the same interaction pattern with anti-apoptotic Bcl-2 family members (A) Bmf S and Bmf CUG were overexpressed simultaneously in 293T cells together with FLAG-tagged versions of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, BHRF1 or KS-Bcl2. Expression of transgene-derived protein was confirmed by immunoblotting using anti-FLAG- or anti-Bmf-specific mAbs (# = marker labeling). (B) Immunoprecipitation was performed using anti-FLAG-M2 mAb. Immune complexes were separated by SDS-PAGE on 4-20% Tris-glycine gels. After electroblotting, nitrocellulose membranes were subjected to immunoblotting using the anti-Bmf mAb (17A9). Membranes were stripped and reprobed with a rat-anti-FLAG antibody. One out of three independent experiments yielding similar results is shown. (* short exposure, ** long exposure)
Figure Legend Snippet: Bmf S and Bmf CUG show the same interaction pattern with anti-apoptotic Bcl-2 family members (A) Bmf S and Bmf CUG were overexpressed simultaneously in 293T cells together with FLAG-tagged versions of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, BHRF1 or KS-Bcl2. Expression of transgene-derived protein was confirmed by immunoblotting using anti-FLAG- or anti-Bmf-specific mAbs (# = marker labeling). (B) Immunoprecipitation was performed using anti-FLAG-M2 mAb. Immune complexes were separated by SDS-PAGE on 4-20% Tris-glycine gels. After electroblotting, nitrocellulose membranes were subjected to immunoblotting using the anti-Bmf mAb (17A9). Membranes were stripped and reprobed with a rat-anti-FLAG antibody. One out of three independent experiments yielding similar results is shown. (* short exposure, ** long exposure)

Techniques Used: Expressing, Derivative Assay, Marker, Labeling, Immunoprecipitation, SDS Page

Related Articles

SDS Page:

Article Title: Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells
Article Snippet: In addition, no detectable signal for PSGL-1, CD43, CD44, HECA-452, or E-Ig was observed in the lysate after the removal of all 3 glycoproteins, suggesting that each IP was performed to completion and that no other glycoprotein(s) contributed to E-selectin ligand activity in these immunoprecipitated samples. .. For SDS-PAGE and Western blots of quantified protein lysates or of immunoprecipitated protein, samples were diluted in reducing sample buffer, boiled, and then separated on 4% to 20% or 7.5% Criterion Tris-HCl SDS-PAGE gels (Bio-Rad) as described previously. .. SDS-PAGE–resolved proteins were transferred to Sequi-blot PVDF membrane (Bio-Rad), and the membrane was blocked with milk/Tris-buffered saline.

Article Title: Proteomic characterization of oyster shell organic matrix proteins (OMP)
Article Snippet: Protein separation by one dimensional gel electrophoresis (1-DE) 40 μg of shell protein samples were deglycosylated using a set of enzymes kit (New England BioLabs) [ ]. .. The deglycosylated mixture was further complemented with 1% bromophenol blue, β-mercaptoethanol, heated to 95oC for 5 min., 40 μg of protein from each was loaded in 10% SDS-PAGE Criterion Tris-HCl gels (Bio-rad), run at room temperature at 80 V for the first 15 min., then at 150 V for the next 40 min., stained with the coomassie brilliant blue G-250 (CBB) and gels were scanned at an optical resolution of 400 dpi using the GS-800 densitometer (Bio-Rad, Hercules, CA, USA). .. Protein separation by two dimensional gel electrophoresis (2-DE) Protein fractions were cleaned using a ReadyPrep Protein 2-DE purification kit (BioRad), 2-DE rehydration/sample buffer was added to the protein the quantification using The RC DC Protein Assay.

Article Title: CK1α Collaborates with DOUBLETIME to Regulate PERIOD Function in the Drosophila Circadian Clock
Article Snippet: Fly head extracts for Western blotting were homogenized using motorized pestle using RBS (20 m m HEPES pH 7.5, 50 m m KCl, 10% glycerol, 2 m m EDTA, 1% Triton X-100, 0.4% NP-40, 1 m m DTT, 0.5 m m PMSF, 0.01 mg/ml aprotinin, 0.005 mg/ml leupeptin, and 0.001 mg/ml pepstatin A). .. Proteins were resolved using 5% Criterion Tris-HCl SDS-PAGE gels (Bio-Rad) and loading was assessed using α-HSP70 (Sigma-Aldrich). .. To classify and quantify hypophosphorylated versus hyperphosphorylated PER isoforms by Western blotting, we used PER isoforms present at ZT8-16 in TUG control flies as reference because PER has been shown to be hypophosphorylated at those circadian time points ( ).

Article Title: Heterologous Expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding
Article Snippet: .. For both total and soluble protein, 5 μl (~0.6–1 μg of total protein) was subjected to electrophoresis on 8%−16% Tris-HCl SDS-PAGE Criterion gels (Bio-Rad), after which the protein in the gel was transferred to nitrocellulose membranes by electrophoresis at 100V, 200mA for 2 hrs at 4°C in Transfer Buffer (0.025 M Tris base, 0.192 M glycine, 0.02% SDS, 20% methanol). .. Membranes were rinsed in PBS (10 mM Sodium Phosphate pH 7.8, 150 mM NaCl), blocked in PBS containing 5% Calf Serum and 0.1% Tween overnight, rinsed twice for 5 min in PBS with 0.1% Tween, incubated with Rat anti-HA high affinity monoclonal antibody clone 3F10 (Roche 1 867 423) at 1: 3,000 dilution in PBS containing 5% Calf Serum for 2 hrs, washed five times with PBS containing 5% Calf Serum and 0.1% Tween.

Western Blot:

Article Title: Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells
Article Snippet: In addition, no detectable signal for PSGL-1, CD43, CD44, HECA-452, or E-Ig was observed in the lysate after the removal of all 3 glycoproteins, suggesting that each IP was performed to completion and that no other glycoprotein(s) contributed to E-selectin ligand activity in these immunoprecipitated samples. .. For SDS-PAGE and Western blots of quantified protein lysates or of immunoprecipitated protein, samples were diluted in reducing sample buffer, boiled, and then separated on 4% to 20% or 7.5% Criterion Tris-HCl SDS-PAGE gels (Bio-Rad) as described previously. .. SDS-PAGE–resolved proteins were transferred to Sequi-blot PVDF membrane (Bio-Rad), and the membrane was blocked with milk/Tris-buffered saline.

Immunoprecipitation:

Article Title: Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells
Article Snippet: In addition, no detectable signal for PSGL-1, CD43, CD44, HECA-452, or E-Ig was observed in the lysate after the removal of all 3 glycoproteins, suggesting that each IP was performed to completion and that no other glycoprotein(s) contributed to E-selectin ligand activity in these immunoprecipitated samples. .. For SDS-PAGE and Western blots of quantified protein lysates or of immunoprecipitated protein, samples were diluted in reducing sample buffer, boiled, and then separated on 4% to 20% or 7.5% Criterion Tris-HCl SDS-PAGE gels (Bio-Rad) as described previously. .. SDS-PAGE–resolved proteins were transferred to Sequi-blot PVDF membrane (Bio-Rad), and the membrane was blocked with milk/Tris-buffered saline.

Protein Concentration:

Article Title: Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants [OPEN]
Article Snippet: The insoluble debris was removed by centrifugation. .. The protein concentration was quantified by Bradford assays, and 10 μg of total protein for each sample was run on a 12.5% Criterion Tris-HCl protein gel (3450014, Bio-Rad). .. Following electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane and then blocked overnight with 5% skim milk in TBS plus 0.1% (v/v) Tween 20 (TBST).

Article Title: Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice
Article Snippet: Frozen muscles ( n = 8 per group) were homogenized in 19 (gastrocnemius and heart) or 29 (soleus) volumes (wt/vol) of homogenization buffer (50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml soybean trypsin inhibitor; pH 7.4) and clarified by centrifugation at 800 g and 4°C. .. Protein concentration of the homogenates was determined (DC Protein Assay, Biorad, Hercules, CA), and equal muscle protein was loaded on Tris·HCl gels (Bio-Rad Criterion System, Hercules, CA). .. Proteins were then transferred to polyvinylidene difluoride membranes, which were subsequently stained with Ponceau S to verify even transfer and protein loading across lanes.

DC Protein Assay:

Article Title: Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice
Article Snippet: Frozen muscles ( n = 8 per group) were homogenized in 19 (gastrocnemius and heart) or 29 (soleus) volumes (wt/vol) of homogenization buffer (50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml soybean trypsin inhibitor; pH 7.4) and clarified by centrifugation at 800 g and 4°C. .. Protein concentration of the homogenates was determined (DC Protein Assay, Biorad, Hercules, CA), and equal muscle protein was loaded on Tris·HCl gels (Bio-Rad Criterion System, Hercules, CA). .. Proteins were then transferred to polyvinylidene difluoride membranes, which were subsequently stained with Ponceau S to verify even transfer and protein loading across lanes.

Polyacrylamide Gel Electrophoresis:

Article Title: MIR-206 regulates connexin43 expression during skeletal muscle development
Article Snippet: Protein extracts were prepared in RIPA buffer [1% NP-40, 1% Deoxycholate, 0.1% SDS, 500 mM Tris, 150 mM NaCl, 1 mM PMSF, 1× Protease Inhibitor Cocktail (Roche)]. .. 10 μg of total protein was separated on precast 12% Tris–HCl PAGE gels (BioRad) and electrotransferred to PVDF membranes. .. Primary antibodies binding to Cx43 (Sigma, rabbit polyclonal, 1:1000 dilution), myogenin (Sigma, rabbit polyclonal, 1:1000), α-tubulin (Sigma, mouse monoclonal, 1:1000) were detected with HRP-conjugated anti-rabbit (Sigma) or HRP-conjugated anti-mouse antibodies (Sigma) and the ECLplus Western Blotting Detection Kit (Amersham).

Article Title: UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation
Article Snippet: Loading buffer (2X) was added to cell lysates, consisting of 125 mM Tris(hydroxymethyl)aminomethane HCl (Research Organics, Cleveland, OH, USA), 4% sodium dodecylsulfate (SDS) (Research Organics, Cleveland, OH, USA), 20% glycerol, and 0.02% bromophenol blue. .. Samples were subsequently boiled and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-HCl Criterion gels (BioRad, Hercules, CA, USA).Protein was transferred to fluorescent-polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). .. Membranes were next blocked in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h, then incubated in Odyssey blocking buffer containing 0.2% Tween 20 (polyoxyethylene-sorbitan monolaurate) and mouse-anti-phospho-ERK 1/2 (Thr202 /Tyr204 ) and rabbit-anti-ERK 1/2 (Cell Signaling Technology, Boston, MA, USA) primary antibodies.

Nucleic Acid Electrophoresis:

Article Title: UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation
Article Snippet: Loading buffer (2X) was added to cell lysates, consisting of 125 mM Tris(hydroxymethyl)aminomethane HCl (Research Organics, Cleveland, OH, USA), 4% sodium dodecylsulfate (SDS) (Research Organics, Cleveland, OH, USA), 20% glycerol, and 0.02% bromophenol blue. .. Samples were subsequently boiled and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-HCl Criterion gels (BioRad, Hercules, CA, USA).Protein was transferred to fluorescent-polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). .. Membranes were next blocked in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h, then incubated in Odyssey blocking buffer containing 0.2% Tween 20 (polyoxyethylene-sorbitan monolaurate) and mouse-anti-phospho-ERK 1/2 (Thr202 /Tyr204 ) and rabbit-anti-ERK 1/2 (Cell Signaling Technology, Boston, MA, USA) primary antibodies.

Staining:

Article Title: Proteomic characterization of oyster shell organic matrix proteins (OMP)
Article Snippet: Protein separation by one dimensional gel electrophoresis (1-DE) 40 μg of shell protein samples were deglycosylated using a set of enzymes kit (New England BioLabs) [ ]. .. The deglycosylated mixture was further complemented with 1% bromophenol blue, β-mercaptoethanol, heated to 95oC for 5 min., 40 μg of protein from each was loaded in 10% SDS-PAGE Criterion Tris-HCl gels (Bio-rad), run at room temperature at 80 V for the first 15 min., then at 150 V for the next 40 min., stained with the coomassie brilliant blue G-250 (CBB) and gels were scanned at an optical resolution of 400 dpi using the GS-800 densitometer (Bio-Rad, Hercules, CA, USA). .. Protein separation by two dimensional gel electrophoresis (2-DE) Protein fractions were cleaned using a ReadyPrep Protein 2-DE purification kit (BioRad), 2-DE rehydration/sample buffer was added to the protein the quantification using The RC DC Protein Assay.

Electrophoresis:

Article Title: Heterologous Expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding
Article Snippet: .. For both total and soluble protein, 5 μl (~0.6–1 μg of total protein) was subjected to electrophoresis on 8%−16% Tris-HCl SDS-PAGE Criterion gels (Bio-Rad), after which the protein in the gel was transferred to nitrocellulose membranes by electrophoresis at 100V, 200mA for 2 hrs at 4°C in Transfer Buffer (0.025 M Tris base, 0.192 M glycine, 0.02% SDS, 20% methanol). .. Membranes were rinsed in PBS (10 mM Sodium Phosphate pH 7.8, 150 mM NaCl), blocked in PBS containing 5% Calf Serum and 0.1% Tween overnight, rinsed twice for 5 min in PBS with 0.1% Tween, incubated with Rat anti-HA high affinity monoclonal antibody clone 3F10 (Roche 1 867 423) at 1: 3,000 dilution in PBS containing 5% Calf Serum for 2 hrs, washed five times with PBS containing 5% Calf Serum and 0.1% Tween.

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    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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    Bio-Rad criterion tris hcl precast sds page gels
    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 <t>SDS-PAGE</t> 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.
    Criterion Tris Hcl Precast Sds Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, 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.

    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: 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.

    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: 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