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    Bio-Rad tris hcl ready gels
    Tris Hcl Ready Gels, 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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    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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    • sds page ready gels  (Bio-Rad)
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    Bio-Rad sds page ready gels
    Schematic overview of the experimental workflow for partial metabolic labeling. Mice with full-thickness burn injury and sham-treated controls were maintained on diets containing (isopropyl- 2 H 7 ) or natural L-Leu for 7 days. Liver tissue from the 2 groups of animals (n=3) were harvested and homogenized on Day 7. The homogenates were sonicated and subjected to 3 freeze-thaw cycles. Hydrophobic components were removed by chloroform extraction. A: corrected relative quantification for partially labeled SILAM. The intensity of unlabeled parent ion from burned animals (obtained by enrichment C1) was subtracted from the MS ion intensity of the corresponding light peptide after mixing the tissue samples. Unambiguous characterizations of partially metabolically labeled liver Akt1/PKBα were achieved with the isotope dilution method and MS/MS sequence information (A2, B2 and C2). B: relative quantification of liver Akt1/PKBα after burn injury. A mixture of the exact amounts (2 g each, isotope dilution) of liver from burned (heavy isotope labeled) and sham-treated (light isotope labeled) mice was homogenized and immunoprecipitated. The free cysteine residues were acetylated with iodoacetyl-LC-biotin at room temperature for 15 min followed by quenching with 2-mercaptoethanol. The beads were then heated at 95°C for 5 min and kept at room temperature for 30 min prior to <t>SDS-PAGE.</t> Biotinylated peptides, including the kinase loop peptide 290 ITDFGLCK 297 , were captured with immobilized monomeric avidin beads. The control peptide, 252 FYGAEIVSALDYLHSEK 268 located just outside of the kinase loop (for assessment of kinase protein level after injury) was obtained from supernatant (B1). Unambiguous confirmations of the peptides and the expected labelings were obtained from singly charged y ions (B2). C: Akt1/PKBα enrichment determination. Akt1/PKBα was immunoprecipitated and free cysteine was biotinylated as described above. Doubly and triply charged tryptic peptides were analyzed with nano-LC interfaced with Q-TOF micro tandem mass spectrometry (C1). Partially labeled parent ions with MS difference of 3.5 Da (doubly charged under ESI) were confirmed via their singly charged heavy y ions with MS difference of 7.0 Da (C2). The monoisotopic parent ion ratio of non-labeled (M+2H + ) and labeled (M+7+2H + ) peptides represents the metabolic labeling efficiency of one instance of (isopropyl- 2 H 7 )-L-Leu incorporation after burn injury. This is an updated proteomic version of classical isotope internal standard characterization.
    Sds Page Ready Gels, 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
    https://www.bioz.com/result/sds page ready gels/product/Bio-Rad
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    acrylamide tris hcl ready sds page gels  (Bio-Rad)
    97
    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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    linear gradient sds page  (Bio-Rad)
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    Bio-Rad linear gradient sds page
    Total nuclear HDAC activity, and HDAC2 protein level and activity are upregulated in mdx mice. Total nuclear HDAC activity of adductors (A), TA (B) and heart (C) were measured by a fluorescent deacetylation assay in mdx 4CV mice compared with controls (5-month-old males, n =5; upregulation in mdx 4CV 2.69- and 1.97-fold in the adductors and TA, respectively). (D) Total nuclear HDAC activity in control mice. (E,F) Western blots of adductor, TA and heart of mdx 4CV mice and controls, analyzed with anti-HDAC2 antibody and quantified normalizing to total nuclear protein ( supplementary material Fig. S2 ; upregulation in mdx 4CV 4.34- and 4.6-fold in the adductors and TA, respectively). (G) Nuclear HDAC2 was immunoprecipitated with anti-HDAC2. The bound proteins were separated by <t>SDS-PAGE</t> and analyzed by western blotting with anti-HDAC2 antibody. (H–K) Immunoprecipitated nuclear HDAC2 activity was determined in adductor (H), TA (I) and heart (J) of mdx 4CV and control mice (upregulation in mdx 4CV 2.52-, 1.95- and 1.15-fold, respectively), and tricep (K) of mdx mice compared with control (2.76-fold, 4.5-month-old males, n =3); * P
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    Schematic overview of the experimental workflow for partial metabolic labeling. Mice with full-thickness burn injury and sham-treated controls were maintained on diets containing (isopropyl- 2 H 7 ) or natural L-Leu for 7 days. Liver tissue from the 2 groups of animals (n=3) were harvested and homogenized on Day 7. The homogenates were sonicated and subjected to 3 freeze-thaw cycles. Hydrophobic components were removed by chloroform extraction. A: corrected relative quantification for partially labeled SILAM. The intensity of unlabeled parent ion from burned animals (obtained by enrichment C1) was subtracted from the MS ion intensity of the corresponding light peptide after mixing the tissue samples. Unambiguous characterizations of partially metabolically labeled liver Akt1/PKBα were achieved with the isotope dilution method and MS/MS sequence information (A2, B2 and C2). B: relative quantification of liver Akt1/PKBα after burn injury. A mixture of the exact amounts (2 g each, isotope dilution) of liver from burned (heavy isotope labeled) and sham-treated (light isotope labeled) mice was homogenized and immunoprecipitated. The free cysteine residues were acetylated with iodoacetyl-LC-biotin at room temperature for 15 min followed by quenching with 2-mercaptoethanol. The beads were then heated at 95°C for 5 min and kept at room temperature for 30 min prior to SDS-PAGE. Biotinylated peptides, including the kinase loop peptide 290 ITDFGLCK 297 , were captured with immobilized monomeric avidin beads. The control peptide, 252 FYGAEIVSALDYLHSEK 268 located just outside of the kinase loop (for assessment of kinase protein level after injury) was obtained from supernatant (B1). Unambiguous confirmations of the peptides and the expected labelings were obtained from singly charged y ions (B2). C: Akt1/PKBα enrichment determination. Akt1/PKBα was immunoprecipitated and free cysteine was biotinylated as described above. Doubly and triply charged tryptic peptides were analyzed with nano-LC interfaced with Q-TOF micro tandem mass spectrometry (C1). Partially labeled parent ions with MS difference of 3.5 Da (doubly charged under ESI) were confirmed via their singly charged heavy y ions with MS difference of 7.0 Da (C2). The monoisotopic parent ion ratio of non-labeled (M+2H + ) and labeled (M+7+2H + ) peptides represents the metabolic labeling efficiency of one instance of (isopropyl- 2 H 7 )-L-Leu incorporation after burn injury. This is an updated proteomic version of classical isotope internal standard characterization.

    Journal: International Journal of Molecular Medicine

    Article Title: SILAM for quantitative proteomics of liver Akt1/PKB? after burn injury

    doi: 10.3892/ijmm.2011.861

    Figure Lengend Snippet: Schematic overview of the experimental workflow for partial metabolic labeling. Mice with full-thickness burn injury and sham-treated controls were maintained on diets containing (isopropyl- 2 H 7 ) or natural L-Leu for 7 days. Liver tissue from the 2 groups of animals (n=3) were harvested and homogenized on Day 7. The homogenates were sonicated and subjected to 3 freeze-thaw cycles. Hydrophobic components were removed by chloroform extraction. A: corrected relative quantification for partially labeled SILAM. The intensity of unlabeled parent ion from burned animals (obtained by enrichment C1) was subtracted from the MS ion intensity of the corresponding light peptide after mixing the tissue samples. Unambiguous characterizations of partially metabolically labeled liver Akt1/PKBα were achieved with the isotope dilution method and MS/MS sequence information (A2, B2 and C2). B: relative quantification of liver Akt1/PKBα after burn injury. A mixture of the exact amounts (2 g each, isotope dilution) of liver from burned (heavy isotope labeled) and sham-treated (light isotope labeled) mice was homogenized and immunoprecipitated. The free cysteine residues were acetylated with iodoacetyl-LC-biotin at room temperature for 15 min followed by quenching with 2-mercaptoethanol. The beads were then heated at 95°C for 5 min and kept at room temperature for 30 min prior to SDS-PAGE. Biotinylated peptides, including the kinase loop peptide 290 ITDFGLCK 297 , were captured with immobilized monomeric avidin beads. The control peptide, 252 FYGAEIVSALDYLHSEK 268 located just outside of the kinase loop (for assessment of kinase protein level after injury) was obtained from supernatant (B1). Unambiguous confirmations of the peptides and the expected labelings were obtained from singly charged y ions (B2). C: Akt1/PKBα enrichment determination. Akt1/PKBα was immunoprecipitated and free cysteine was biotinylated as described above. Doubly and triply charged tryptic peptides were analyzed with nano-LC interfaced with Q-TOF micro tandem mass spectrometry (C1). Partially labeled parent ions with MS difference of 3.5 Da (doubly charged under ESI) were confirmed via their singly charged heavy y ions with MS difference of 7.0 Da (C2). The monoisotopic parent ion ratio of non-labeled (M+2H + ) and labeled (M+7+2H + ) peptides represents the metabolic labeling efficiency of one instance of (isopropyl- 2 H 7 )-L-Leu incorporation after burn injury. This is an updated proteomic version of classical isotope internal standard characterization.

    Article Snippet: SDS-PAGE ready gels (12% Tris-HCl, #161–1102), Laemmli sample buffer (#161–0737) and Coomassie Brilliant Blue R-250 (#161–0436) were obtained from Bio-Rad.

    Techniques: Labeling, Mouse Assay, Sonication, Mass Spectrometry, Metabolic Labelling, Isotope Dilution, Sequencing, Immunoprecipitation, SDS Page, Avidin-Biotin Assay

    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

    A 16.5% Tris-Tricine SDS-PAGE Gel stained with Coomassie G-250 using the Laemmli buffer system (1.0 M Tris, pH 8.45). Std, Polypeptide Standards. A comparison of ( A ) casein versus. ( B ) CPP is presented to the show CPP from the tryptic digested casein.

    Journal: Antioxidants

    Article Title: Antioxidant Properties of Casein Phosphopeptides (CPP) and Maillard-Type Conjugated Products

    doi: 10.3390/antiox9080648

    Figure Lengend Snippet: A 16.5% Tris-Tricine SDS-PAGE Gel stained with Coomassie G-250 using the Laemmli buffer system (1.0 M Tris, pH 8.45). Std, Polypeptide Standards. A comparison of ( A ) casein versus. ( B ) CPP is presented to the show CPP from the tryptic digested casein.

    Article Snippet: Molecular WeightCasein and CPP were analyzed by electrophoresis using 16.5% acrylamide Tris-Tricine (N-tris [hydroxymethyl] methyl glycine) SDS (sodium dodecyl sulfate) ready gel according to Laemmli [ ], and a Mini-Protean I Mini-Cell slab gel electrophoresis unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA).

    Techniques: SDS Page, Staining

    Total nuclear HDAC activity, and HDAC2 protein level and activity are upregulated in mdx mice. Total nuclear HDAC activity of adductors (A), TA (B) and heart (C) were measured by a fluorescent deacetylation assay in mdx 4CV mice compared with controls (5-month-old males, n =5; upregulation in mdx 4CV 2.69- and 1.97-fold in the adductors and TA, respectively). (D) Total nuclear HDAC activity in control mice. (E,F) Western blots of adductor, TA and heart of mdx 4CV mice and controls, analyzed with anti-HDAC2 antibody and quantified normalizing to total nuclear protein ( supplementary material Fig. S2 ; upregulation in mdx 4CV 4.34- and 4.6-fold in the adductors and TA, respectively). (G) Nuclear HDAC2 was immunoprecipitated with anti-HDAC2. The bound proteins were separated by SDS-PAGE and analyzed by western blotting with anti-HDAC2 antibody. (H–K) Immunoprecipitated nuclear HDAC2 activity was determined in adductor (H), TA (I) and heart (J) of mdx 4CV and control mice (upregulation in mdx 4CV 2.52-, 1.95- and 1.15-fold, respectively), and tricep (K) of mdx mice compared with control (2.76-fold, 4.5-month-old males, n =3); * P

    Journal: Disease Models & Mechanisms

    Article Title: Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy

    doi: 10.1242/dmm.013631

    Figure Lengend Snippet: Total nuclear HDAC activity, and HDAC2 protein level and activity are upregulated in mdx mice. Total nuclear HDAC activity of adductors (A), TA (B) and heart (C) were measured by a fluorescent deacetylation assay in mdx 4CV mice compared with controls (5-month-old males, n =5; upregulation in mdx 4CV 2.69- and 1.97-fold in the adductors and TA, respectively). (D) Total nuclear HDAC activity in control mice. (E,F) Western blots of adductor, TA and heart of mdx 4CV mice and controls, analyzed with anti-HDAC2 antibody and quantified normalizing to total nuclear protein ( supplementary material Fig. S2 ; upregulation in mdx 4CV 4.34- and 4.6-fold in the adductors and TA, respectively). (G) Nuclear HDAC2 was immunoprecipitated with anti-HDAC2. The bound proteins were separated by SDS-PAGE and analyzed by western blotting with anti-HDAC2 antibody. (H–K) Immunoprecipitated nuclear HDAC2 activity was determined in adductor (H), TA (I) and heart (J) of mdx 4CV and control mice (upregulation in mdx 4CV 2.52-, 1.95- and 1.15-fold, respectively), and tricep (K) of mdx mice compared with control (2.76-fold, 4.5-month-old males, n =3); * P

    Article Snippet: HDAC2 protein level Nuclear protein fractions (method described above) were separated using 4–20% linear gradient SDS-PAGE (Tris-HCl Ready Gel, Bio-Rad, Hercules, CA) and transferred to polyvinylidene fluoride (PVDF) membranes with a wet transfer system (Bio-Rad).

    Techniques: Activity Assay, Mouse Assay, Western Blot, Immunoprecipitation, SDS Page