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  • 99
    Thermo Fisher sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to <t>SDS-PAGE,</t> and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by <t>SDS-PAGE</t> and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sds polyacrylamide gel electrophoresis
    Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were <t>delipidated,</t> and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% <t>SDS-polyacrylamide</t> gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    Protein analysis of M. tuberculosis. (A) <t>SDS-PAGE</t> analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 12745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad sds page
    Protein analysis of M. tuberculosis. (A) <t>SDS-PAGE</t> analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.
    Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 4860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore sds page
    Protein analysis of M. tuberculosis. (A) <t>SDS-PAGE</t> analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.
    Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 4072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds page electrophoresis
    A) Image of cellular targets of EPO in mouse brain separated by <t>SDS-PAGE</t> in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining
    Sds Page Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher polyacrylamide gel electrophoresis
    A) Image of cellular targets of EPO in mouse brain separated by <t>SDS-PAGE</t> in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining
    Polyacrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad gradient sds page gels
    p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in <t>SDS-PAGE</t> gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P
    Gradient Sds Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher sds polyacrylamide gel electrophoresis
    <t>SDS-PAGE</t> of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds page gel
    Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using <t>SDS-PAGE.</t> Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.
    Sds Page Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher bis tris sds page gels
    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates <t>(SDS</t> fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing <t>SDS/PAGE</t> and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.
    Bis Tris Sds Page Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc sodium dodecyl sulfate polyacrylamide gel electrophoresis
    TCR-mediated signaling in TRIM-deficient CD4 + T cells. (A) TRIM expression in T cells. Postnuclear lysates were prepared from freshly isolated thymocytes, lymph node cells (LN), or purified CD4 + and CD8 + splenocytes, separated on <t>sodium</t> <t>dodecyl</t> <t>sulfate-polyacrylamide</t> <t>gel</t> <t>electrophoresis,</t> and blotted with TRIM04 MAb. The blot was reprobed with anti-Erk1/2 to demonstrate equal loading. Numbers below the blot represent relative TRIM expression compared to the lowest. (B) Global tyrosine phosphorylation and MAP family kinase activation are normal, but Akt phosphorylation is enhanced in TRIM-deficient mice. Purified CD4 + splenic T cells were stimulated for the indicated periods of time with the CD3 MAb 145-2C11. The cells were lysed and processed for Western blot analysis. The blots were probed with anti-phosphospecific antibodies and then stripped and reprobed with anti-Erk1/2, anti-ZAP-70, and anti-Akt to show equal loading. Data are representative of five separate experiments. (C) Analysis of Ca 2+ mobilization. Purified splenic CD4 + cells were loaded with indo-1-AM and incubated with 10 μg/ml of biotinylated anti-CD3 (145-2C11). The arrows indicate the addition of streptavidin and ionomycin (Iono), respectively. (D) TRIM regulation of Akt phosphorylation depends on PI3K activity. Purified CD4 + splenic T cells were stimulated for 2 min with CD3 MAb alone or with CD3 plus CD28 MAbs in the presence or absence of the PI3K inhibitor wortmannin. The level of Akt activation was measured by using a phosphospecific anti-Akt antibody and the equal loading by using an anti-Akt antibody. stim., stimulation.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

    Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

    doi: 10.1128/IAI.00004-09

    Figure Lengend Snippet: Analysis of LPS-Trap-Fc1 to -Fc4. (A) Schematic representation of the mTLR4/mMD-2 fusion proteins LPS-Trap (left top) and LPS-Trap-Fc1 (left bottom and right). (B) Supernatants of LPS-Trap-Fc1-, -Fc2-, -Fc3-, or -Fc4-transfected HEK293T cells were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting for the human IgG-Fc tail (left) or the N-terminal FLAG epitope (right).

    Article Snippet: Samples were separated by 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nupage Bis-Tris gel; Invitrogen) and electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).

    Techniques: Transfection, SDS Page, Western Blot, FLAG-tag

    Membrane association of the rotavirus VP5 proteins. HEK293 cells were transfected with pcDNA4 constructs expressing VP5N248, VP5N248(394R), or VP5N265. Cellular membranes were fractionated by sucrose gradient centrifugation. His-tagged VP5N248, VP5N248(394R), or VP5N265 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using an anti-HisG antibody.

    Journal: Journal of Virology

    Article Title: Discrete Domains within the Rotavirus VP5* Direct Peripheral Membrane Association and Membrane Permeability

    doi: 10.1128/JVI.78.4.2037-2044.2004

    Figure Lengend Snippet: Membrane association of the rotavirus VP5 proteins. HEK293 cells were transfected with pcDNA4 constructs expressing VP5N248, VP5N248(394R), or VP5N265. Cellular membranes were fractionated by sucrose gradient centrifugation. His-tagged VP5N248, VP5N248(394R), or VP5N265 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using an anti-HisG antibody.

    Article Snippet: Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using anti-HisG primary antibody (Invitrogen).

    Techniques: Transfection, Construct, Expressing, Gradient Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot

    Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Various expression patterns of VP7 in transfected cells. (A) Purification of VP7 with Ni 2 + column chromatography and detection by SDS-PAGE and Western blotting. Primary antibody was 6*His antibody. M. Protein molecular mass marker; 1–7: The purified protein eluted by 10, 20, 50, 100, 200, 300, 400 mmol/L of imidazole solution respectively. (B) Purification of VP7 detected by Western blotting. Primary antibody was VP7 antibody.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Expressing, Transfection, Purification, Column Chromatography, SDS Page, Western Blot, Marker

    Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Journal: Gene

    Article Title: Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

    doi: 10.1016/j.gene.2017.06.048

    Figure Lengend Snippet: Construction of pET-28a-S7 vector and identification of recombinant VP7 and prepared polyclonal VP7 antibody. (A) Electrophoretogram. (B) SDS-PAGE and Western blotting.

    Article Snippet: The proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting ( B), using a primary antibody of rabbit anti-6 × His (Sigma) and a secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma).

    Techniques: Positron Emission Tomography, Plasmid Preparation, Recombinant, SDS Page, Western Blot

    Transforming activity of a PH domain-minus derivative of Dbs is restored by the addition of a plasma membrane-targeting signal from H-Ras. (A) The domain structure of the full-length Dbs protein is illustrated in the upper line (DH, Dbl homology domain; PH, pleckstrin homology domain; SH3, Src homology 3 domain), and the lines below indicate the regions of the protein included in predicted translation products of the cDNA derivatives. The numbers refer to amino acid positions in the full-length protein. The Dbs-HA7 derivative is fused to the H-Ras COOH-terminal plasma membrane-targeting sequence that includes the CAAX tetrapeptide isoprenylation signal (designated CAAX). All derivatives were fused at their NH 2 termini to an HA epitope tag. The values at the right indicate the transforming activity of the different derivatives in NIH 3T3 cell focus formation assays. The data shown are representative of three independent assays done on triplicate plates. (B) Expression of HA epitope-tagged Dbs proteins. Epitope-tagged proteins were expressed transiently in 293 cells (left-hand lanes) or stably in NIH 3T3 cells (right-hand lanes) and detected by Western blotting with an anti-HA epitope antibody. (C) Subcellular localization of PH domain variants of Dbs. HA epitope-tagged proteins were expressed stably in NIH 3T3 cells. The cells were lysed and fractionated at 100,000 × g . Thirty micrograms of protein from total (T), soluble (S), and particulate (P) fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein expression was determined with the anti-HA epitope antibody (BabCo). The relative amounts of protein in particulate and soluble fractions (%) were determined with a PhosphorImager (Molecular Dynamics).

    Journal: Molecular and Cellular Biology

    Article Title: Dependence of Dbl and Dbs Transformation on MEK and NF-?B Activation

    doi:

    Figure Lengend Snippet: Transforming activity of a PH domain-minus derivative of Dbs is restored by the addition of a plasma membrane-targeting signal from H-Ras. (A) The domain structure of the full-length Dbs protein is illustrated in the upper line (DH, Dbl homology domain; PH, pleckstrin homology domain; SH3, Src homology 3 domain), and the lines below indicate the regions of the protein included in predicted translation products of the cDNA derivatives. The numbers refer to amino acid positions in the full-length protein. The Dbs-HA7 derivative is fused to the H-Ras COOH-terminal plasma membrane-targeting sequence that includes the CAAX tetrapeptide isoprenylation signal (designated CAAX). All derivatives were fused at their NH 2 termini to an HA epitope tag. The values at the right indicate the transforming activity of the different derivatives in NIH 3T3 cell focus formation assays. The data shown are representative of three independent assays done on triplicate plates. (B) Expression of HA epitope-tagged Dbs proteins. Epitope-tagged proteins were expressed transiently in 293 cells (left-hand lanes) or stably in NIH 3T3 cells (right-hand lanes) and detected by Western blotting with an anti-HA epitope antibody. (C) Subcellular localization of PH domain variants of Dbs. HA epitope-tagged proteins were expressed stably in NIH 3T3 cells. The cells were lysed and fractionated at 100,000 × g . Thirty micrograms of protein from total (T), soluble (S), and particulate (P) fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein expression was determined with the anti-HA epitope antibody (BabCo). The relative amounts of protein in particulate and soluble fractions (%) were determined with a PhosphorImager (Molecular Dynamics).

    Article Snippet: The protein concentrations of the total, cytosolic, and membrane fractions were determined with a biciuchoninic acid protein assay kit (Pierce) with 30 μg of protein for each fraction, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore), and probed with anti-HA epitope antibody (BabCo).

    Techniques: Activity Assay, Sequencing, Expressing, Stable Transfection, Western Blot, Polyacrylamide Gel Electrophoresis

    Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Incubation

    Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Isolation, SDS Page, Western Blot

    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page

    Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Software, Expressing, SDS Page, Western Blot, Standard Deviation

    Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page, Standard Deviation

    Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Journal: PLoS ONE

    Article Title: Insights on the Evolution of Prolyl 3-Hydroxylation Sites from Comparative Analysis of Chicken and Xenopus Fibrillar Collagens

    doi: 10.1371/journal.pone.0019336

    Figure Lengend Snippet: Isolated collagen species from chicken and xenopus on 6% SDS-PAGE. A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.

    Article Snippet: Protein samples were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue G-250 (Sigma-Aldrich) .

    Techniques: Isolation, SDS Page

    Gastrin induced phosphorylation of protein kinase C (PKC)-δ. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, 30, or 60 minutes and cell lysates were then obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to membranes. Membranes were reacted with rabbit antihuman-phospho-PKC-δ (Thr505) polyclonal antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system.

    Journal: Gut

    Article Title: Gastrin activates nuclear factor ?B (NF?B) through a protein kinase C dependent pathway involving NF?B inducing kinase, inhibitor ?B (I?B) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor

    doi:

    Figure Lengend Snippet: Gastrin induced phosphorylation of protein kinase C (PKC)-δ. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, 30, or 60 minutes and cell lysates were then obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to membranes. Membranes were reacted with rabbit antihuman-phospho-PKC-δ (Thr505) polyclonal antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system.

    Article Snippet: Aliquots containing 50 μg of total protein were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (5–20% gradient gels), and proteins were transferred to polyvinylidine difluoride membranes (Immobilon; Millipore, Bedford, Massachusetts, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation

    Gastrin induced degradation of inhibitor κB (IκB)-α. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, or 30 minutes and cell lysates were obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidine difluoride membranes. Membranes were reacted with rabbit antihuman-IκB-α antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system. Quantitated signals were determined by densitometry.

    Journal: Gut

    Article Title: Gastrin activates nuclear factor ?B (NF?B) through a protein kinase C dependent pathway involving NF?B inducing kinase, inhibitor ?B (I?B) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor

    doi:

    Figure Lengend Snippet: Gastrin induced degradation of inhibitor κB (IκB)-α. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, or 30 minutes and cell lysates were obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidine difluoride membranes. Membranes were reacted with rabbit antihuman-IκB-α antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system. Quantitated signals were determined by densitometry.

    Article Snippet: Aliquots containing 50 μg of total protein were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (5–20% gradient gels), and proteins were transferred to polyvinylidine difluoride membranes (Immobilon; Millipore, Bedford, Massachusetts, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation

    Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Journal: Journal of Bacteriology

    Article Title: Microaerophilic Induction of the Alpha-Crystallin Chaperone Protein Homologue (hspX) mRNA of Mycobacterium tuberculosis

    doi: 10.1128/JB.183.18.5311-5316.2001

    Figure Lengend Snippet: Protein analysis of M. tuberculosis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis stained with Coomassie blue. Cultures were incubated for the indicated times in slowly stirred sealed tubes. AG indicates shaking aerobic incubation for 80 h. The alpha-crystallin homologue is indicated. (B) Immunoblot analysis of the Acr protein. Proteins from either aerobic (AG) or 141-h-old hypoxic cultures were probed with either monoclonal anti-Acr (CS49) or polyclonal anti-URB-1 (RS88-13078) antibody.

    Article Snippet: Twenty micrograms of protein for each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-tricine 16.5% polyacrylamide gel (Bio-Rad).

    Techniques: SDS Page, Staining, Incubation

    Silver-stained SDS-PAGE of pRBC saponin extracts run through four affinity chromatography columns where AGMA1, ISA1, ISA23 or ARGO7 had been immobilized. ( A ) A RBC extract was first loaded, and after the corresponding washing-elution-washing steps ( B ) a pRBC extract was loaded in the same column. The approximate masses (kDa) of the seven bands from the molecular weight marker are indicated in the space between the gels.

    Journal: Pharmaceutics

    Article Title: Polyamidoamine Nanoparticles for the Oral Administration of Antimalarial Drugs

    doi: 10.3390/pharmaceutics10040225

    Figure Lengend Snippet: Silver-stained SDS-PAGE of pRBC saponin extracts run through four affinity chromatography columns where AGMA1, ISA1, ISA23 or ARGO7 had been immobilized. ( A ) A RBC extract was first loaded, and after the corresponding washing-elution-washing steps ( B ) a pRBC extract was loaded in the same column. The approximate masses (kDa) of the seven bands from the molecular weight marker are indicated in the space between the gels.

    Article Snippet: For SDS-polyacrylamide gel electrophoresis (PAGE) analysis, samples were heated at 90 °C for 5 min in an elution buffer, and electrophoresed in 1 mm-thick 12.5% SDS-polyacrylamide gels (Mini Protean II System, Bio-Rad), which were silver-stained as previously described [ ].

    Techniques: Staining, SDS Page, Affinity Chromatography, Molecular Weight, Marker

    Effects of IXD extract on the morphology of submandibular glands and in salivary total protein expression. Notes: ( A ) Hematoxylin and eosin staining was performed on paraffin-embedded submandibular gland tissues from normal and diabetic rats, either treated with water or IXD extract. Magnification=20×, scale bar=100 μm. ( B ) CBB staining showing total protein expression in the saliva of control and diabetic rats, either treated with water or IXD extract. The same volume of saliva was loaded in each well of a 10% SDS-PA gel, and the gel was stained with CBB. The black arrow indicates the molecular size of amylase (55 kDa) present in the saliva. Abbreviations: CBB, Coomassie Brilliant Blue; IXD, Ixeris dentata ; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Journal: Journal of Experimental Pharmacology

    Article Title: Ixeris dentata extract regulates salivary secretion through the activation of aquaporin-5 and prevents diabetes-induced xerostomia

    doi: 10.2147/JEP.S141807

    Figure Lengend Snippet: Effects of IXD extract on the morphology of submandibular glands and in salivary total protein expression. Notes: ( A ) Hematoxylin and eosin staining was performed on paraffin-embedded submandibular gland tissues from normal and diabetic rats, either treated with water or IXD extract. Magnification=20×, scale bar=100 μm. ( B ) CBB staining showing total protein expression in the saliva of control and diabetic rats, either treated with water or IXD extract. The same volume of saliva was loaded in each well of a 10% SDS-PA gel, and the gel was stained with CBB. The black arrow indicates the molecular size of amylase (55 kDa) present in the saliva. Abbreviations: CBB, Coomassie Brilliant Blue; IXD, Ixeris dentata ; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Article Snippet: Thirty µg of total protein were loaded per lane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; BioRad), and transferred to a polyvinylidene fluoride membrane.

    Techniques: Expressing, Staining, SDS Page, Polyacrylamide Gel Electrophoresis

    ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: ) and separated by SDS/PAGE. The glycoproteins and receptors indicated to the Left were identified by WB. For each sample, three replicate gels were developed as follows: one for gB and gD, one for gH and gL, and a third one for αv-integrin and nectin1. ( A ) The complex pulled down by αv STREP β6-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6-integrin lacks gL (lane 1, fuchsia star). In contrast, the complex assembled onto αvβ6-integrin alone (lane 4, green star) or nectin1 alone (lane 2) contains gL. ( B ) The complex assembled on αvβ8-integrin in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β8-integrin lacks gL (lane 8, fuchsia star). ( C ) The complex assembled on αvβ6N1-chimera in cells transfected with the four glycoproteins, gD, gH, gL, and gB, plus nectin1, αv-integrin, and β6N-chimera lacks gL (lane 11, fuchsia star). ( D – F ) WB analysis of lysates from samples A – C ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A – C .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot, Transfection

    , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: , and separated by SDS/PAGE. Glycoproteins and receptors were identified by WB. It can be seen that the complex assembled onto αvβ6-integrin in the presence of nectin1 lacks gL (lane 3, fuchsia star). When gD (lane 4, HLB), gB (lane 8, DHL), or nectin1 (lanes 5, 6, 9, and αβ6) were missing, the complexes contained gL. ( C and D ) WB analysis of lysates from samples A and B ). The insoluble material was pelleted. Ten microliters of the supernatant was subjected to SDS/PAGE and WB with antibodies to the indicated proteins. The remaining 90 μL were used for the pulldown experiments shown in A and B .

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: SDS Page, Western Blot

    Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

    doi: 10.1073/pnas.1506846112

    Figure Lengend Snippet: Block of HSV-1 infection by the neutralizing MAb LP11 to gH prevents the dissociation of gL from virion gH/gL. HSV-1(F) virions were preincubated with MAbs 52S, 53S, LP11, or IgGs. J cells expressing αvβ6-integrin plus nectin1 (N+αβ6), or nectin1 alone (N) were exposed to the preincubated virions. After 30 min, the media were harvested and subjected to SDS/PAGE and WB for gL. The medium of cells exposed to LP11-treated virions lacks gL. Lane HSV to the Left shows the WB reactivity of the indicated virion glycoproteins.

    Article Snippet: Proteins retained by Strep-Tactin Sepharose were separated by SDS polyacrylamide gel electrophoresis (SDS/PAGE), western blotted (WB) with appropriate antibodies, and developed by means of ChemiDoc XRS+ using Image Lab Software (Biorad).

    Techniques: Blocking Assay, Infection, Expressing, SDS Page, Western Blot

    SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Lysine at the C-Terminus of an Odorant-Binding Protein is Involved in Binding Aldehyde Pheromone Components in Two Helicoverpa Species

    doi: 10.1371/journal.pone.0055132

    Figure Lengend Snippet: SDS-PAGE and Western blot of extracts from parts of the body of adults H. armigera and H. assulta . Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers ( M ) are as in Figure 1 .

    Article Snippet: Western Blot Analysis After electrophoretic separation under 14% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), duplicate gels were stained with 0.1% Coomassie blue R250 in 10% acetic acid, 20% ethanol or electroblotted on Trans-Blot nitrocellulose membrane (Bio-Rad Lab) by the procedure of Kyhse-Andersen .

    Techniques: SDS Page, Western Blot, Expressing, Staining, Molecular Weight

    Signalling effects of SP600125. We performed Western blot analysis on tissue lysates obtained from each group of animals. After resolution on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis the subsequent steps are as described in the Materials and methods. Phosphospecific antibodies (Pp38, Pp42/44ERK and PpJNK) were obtained from Cell Signaling, and the remaining antibodies were from Santa Cruz (p42ERK and pJNK), and p38 was from Stressgen Biotechnology Corporation (Stressgen, Victoria). The data are representative of the experiment performed three times. (a,b) p38 and p42Erk; the arrows indicate changes induced by inflammation in the phosphosignal for JNK (c, solid arrows) and reduction of the signal with SP600125 treatment (dashed arrows). The arrowheads indicate that there is a reduction in the expression of the slower migrating JNK (p54) band using this inhibitor. In (d) a representative electromobility shift assay autoradiogram is shown. This shows that there is a marked induction of AP-1 DNA binding with DSS treatment that is attenuated with SP600125.

    Journal: Immunology

    Article Title: The specific JNK inhibitor SP600125 targets tumour necrosis factor-? production and epithelial cell apoptosis in acute murine colitis

    doi: 10.1111/j.1365-2567.2006.02349.x

    Figure Lengend Snippet: Signalling effects of SP600125. We performed Western blot analysis on tissue lysates obtained from each group of animals. After resolution on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis the subsequent steps are as described in the Materials and methods. Phosphospecific antibodies (Pp38, Pp42/44ERK and PpJNK) were obtained from Cell Signaling, and the remaining antibodies were from Santa Cruz (p42ERK and pJNK), and p38 was from Stressgen Biotechnology Corporation (Stressgen, Victoria). The data are representative of the experiment performed three times. (a,b) p38 and p42Erk; the arrows indicate changes induced by inflammation in the phosphosignal for JNK (c, solid arrows) and reduction of the signal with SP600125 treatment (dashed arrows). The arrowheads indicate that there is a reduction in the expression of the slower migrating JNK (p54) band using this inhibitor. In (d) a representative electromobility shift assay autoradiogram is shown. This shows that there is a marked induction of AP-1 DNA binding with DSS treatment that is attenuated with SP600125.

    Article Snippet: From each sample, 25 μg protein was resolved using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis before transferring to nitrocellulose membranes (Bio-Rad).

    Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Expressing, Electro Mobility Shift Assay, Binding Assay

    A) Image of cellular targets of EPO in mouse brain separated by SDS-PAGE in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Recognition and characterization of Erythropoietin binding-proteins in the brain of mice

    doi:

    Figure Lengend Snippet: A) Image of cellular targets of EPO in mouse brain separated by SDS-PAGE in a 12 % gel. Gel was stained by Silver. B)Image of cellular targets of EPO in mouse brain separated by IEF in a 7 cm IPG strip containing nonlinear pH gradient 3–10 followed by two-dimensional gel electrophoresis. Protein detection was by Silver-staining

    Article Snippet: Separation of target proteins on SDS-PAGE Freeze dried elutes, from IP, step were dissolved in 20 μl of 2X SDS sample buffer, samples were incubated in boiling water for 5 min and then subjected to SDS-PAGE electrophoresis (BioRad).

    Techniques: SDS Page, Staining, Electrofocusing, Stripping Membranes, Two-Dimensional Gel Electrophoresis, Electrophoresis, Silver Staining

    Sucrose gradient sedimentation profiles of the NUDA and NUDG proteins. A sample from each of the top nine fractions from the gradient was subjected to SDS-PAGE and Western blotted with antibody against the nudA CDHC or the nudG 8-kD CDLC.

    Journal: The Journal of Cell Biology

    Article Title: The "8-kD" Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

    doi:

    Figure Lengend Snippet: Sucrose gradient sedimentation profiles of the NUDA and NUDG proteins. A sample from each of the top nine fractions from the gradient was subjected to SDS-PAGE and Western blotted with antibody against the nudA CDHC or the nudG 8-kD CDLC.

    Article Snippet: The immunoprecipitate was resuspended in Laemmli sample buffer, subjected to electrophoresis on a 4–20% gradient SDS-PAGE gel (Bio-Rad Laboratories, Richmond, CA) and transferred to an Immobilon-P membrane ( Millipore Corp. , Bedford, MA).

    Techniques: Sedimentation, SDS Page, Western Blot

    p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in SDS-PAGE gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P

    Journal: Cancer research

    Article Title: Mouse homolog of the human TP53 R337H mutation reveals its role in tumorigenesis

    doi: 10.1158/0008-5472.CAN-18-0016

    Figure Lengend Snippet: p53 oligomerization is decreased in p53 334H/H mouse tissues. A , liver tissue lysates were cross-linked with glutaraldehyde, resolved in SDS-PAGE gel, and immunoblotted. Note p53 antibody specificity demonstrated by lack of immunoreactivity in the p53 −/− sample. Protein standards are in kD. Triplicate lanes of each genotype represent liver samples from 3 separate mice. B, fraction of p53 oligomers (T, tetramer; D, dimer; M, monomer) within each lane of immunoblot (A) quantified by densitometry and compared with the respective wild-type oligomer ( n = 3). C , p53 immunoblot of cross-linked liver lysates obtained from mice 6 h after p53 induction by doxorubicin treatment. Duplicate lanes of each genotype represent liver samples from 2 separate mice. D , p53 binding to the p53 response element (p53RE) of p21 in γ-irradiated (γ-IR) mouse liver. ChIP was performed using nonspecific IgG or anti-p53 antibody. p53RE binding is shown relative to wild-type non-specific IgG samples ( n = 3). E, induction of p21 mRNA in the indicated tissues by γ-IR quantified by RT-PCR ( n = 3). Levels were normalized relative to a housekeeping gene TIF . Bone marrow (BM); small intestine. p53 R334 genotypes: wild-type ( R/R ); heterozygous mutant ( R/H ); homozygous mutant ( H/H ); and p53 null ( −/− ). Values are mean ± SD. Compared to wild-type samples within each group, statistical differences were tested by 1-way ANOVA. * P

    Article Snippet: The protein samples were resolved on 4–20% gradient SDS/PAGE gels (Bio-Rad Laboratories) and immunoblotted with anti-p53 mouse monoclonal antibody (#2524, Cell Signaling Technology).

    Techniques: SDS Page, Mouse Assay, Binding Assay, Irradiation, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    SDS-PAGE of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.

    Journal: Brazilian Journal of Microbiology

    Article Title: Isolation and Purification of Complex II from Proteus Mirabilis Strain ATCC 29245

    doi: 10.1590/S1517-83822010005000032

    Figure Lengend Snippet: SDS-PAGE of the Proteus mirabilis strain ATCC 29245. The purified cytochrome b was loaded on 12.5% gel. The gel was stained with commassie brilliant blue after electrophoresis. Lane 1 (molecular mass marker proteins), lane 2 (sample 1, partially purified complex II after ion exchange chromatography) and lane 3, (sample 2, purified complex II). The molecular masses of three subunits of the Proteus mirabilis strain ATCC 29245 complex II were estimated to be 68, 28.5, and 19.5 kDa.

    Article Snippet: In the estimation of the apparent molecular weight by SDS-polyacrylamide gel electrophoresis, a set of protein markers (PAGE ruler pre-stained protein ladder, SM0671 Fermentas) with known molecular weights were used.

    Techniques: SDS Page, Purification, Staining, Electrophoresis, Marker, Ion Exchange Chromatography

    Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response

    doi: 10.3390/ijms21145146

    Figure Lengend Snippet: Co-immunoprecipitation of the selective recruitment of Csk and pSHP-2 to various 1.1b WT CYT constructs following co-crosslinking with 2.6b ITAM CYT . AD293 cells (3 × 10 5 ) co-expressing 2.6b ITAM CYT and the various 1.1b CYT constructs were incubated at 37 °C for 8 min with 3 µm magnetic beads (3 × 10 6 ) opsonized with the indicated mAbs and/or isotype IgG1 (’+’ and ‘−’ indicate the presence and absence of corresponding antibodies on beads, respectively). Cells were immediately lysed on ice for 30 min and then magnetic beads were separated, washed three times, and eluted before separating bead-bound proteins using SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and then probed with α-FLAG and α-HA mAbs ( A ) to verify successful pull-down of the various 1.1b CYT constructs (top membrane) and 2.6b ITAM CYT (bottom membrane). Co-immunoprecipitation of potential effector molecules ( B ) was further examined by probing membranes with α-Csk, α-pSHP-2, α-PTEN, and α-SHIP2 mAbs. For the bottom two panels in ( B ), a whole cell lysate (WCL) sample is included to show that these molecules are present in AD293 lysates. Blots shown are representative examples of three independent experiments.

    Article Snippet: Proteins were separated via 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad) that were probed with an HRP-conjugated α-HA mAb (1:1000) and an HRP-conjugated α-FLAG mAb (1:1000; Thermo Fisher Scientific) at 4 °C overnight to verify pull-down of the various epitope-tagged IpLITR constructs.

    Techniques: Immunoprecipitation, Construct, Expressing, Incubation, Magnetic Beads, SDS Page

    New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: New monoclonal antibodies visualize parkin in ageing human midbrain. (a-c) Characterization of four murine, monoclonal antibodies (of IgG 2 isotype; clone-B, -E, -D, and -G) in three different assays: (a) against recombinant (r-), full-length, untagged, wildtype (WT) human parkin using non-denaturing slot blots (100ng/slot) of original antigen as well as truncated r-parkin 321-465 and full-length, untagged, human r-DJ-1; (b) against human brain lysates (SDS fractions from control and PRKN- linked ARPD cases) using non-denaturing dot blots; and (c) by denaturing SDS/PAGE and Western blotting of extracts from cortical specimens of a control brain and a parkin-deficient ARPD case. Screening by these three methods as well as by cell-based microscopy (using indirect immunofluorescence) revealed specific staining for four anti-parkin clones (-B, -E, -D and -G), which was conformation-dependent for clone-E. List of epitopes within the sequence of human parkin, as recognized by clones -B, -E, -D, and -G and informed by overlapping screening with 7-12 amino acid-long peptides covering full-length, human parkin. Note that the clone E epitope is conformational, comprised of the three regions indicated.

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Recombinant, SDS Page, Western Blot, Microscopy, Immunofluorescence, Staining, Clone Assay, Sequencing

    Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin transitions from a soluble to an aggregated state in adult human midbrain. (a) Representative Western blots of parkin, DJ-1, and LC3B distribution in human cortex, S. nigra (SN) and red nucleus (RN) brain specimens that had been serially fractionated into Tris-NaCl buffer-soluble (TS), Triton X-100-soluble (TX), 2% SDS-soluble (SDS) extracts and the pellet (P) lysed in 30% SDS-containing buffer. Lysates from PRKN -linked Parkinson disease (ARPD) brain and recombinant, human parkin (r-parkin) are included. (b-c) Relative distribution of parkin signal within each fraction for (b) cortex and (c) midbrain grouped by age ranges: young (Y; ≤ 20y; n=13); mid (M, > 20y,

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Western Blot, Recombinant

    Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Journal: bioRxiv

    Article Title: Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

    doi: 10.1101/2020.02.19.953034

    Figure Lengend Snippet: Parkin’s secondary structure is altered by redox stress. (a) Silver staining of r-parkin in soluble (supernatant) and insoluble (pellet) phases following exposure to increasing concentrations of H 2 O 2 (0-2mM) and run under non-reducing conditions. Monomer (*) and high M r weight (HMW) r-parkin species are indicated. (b) Silver stained gel of r-parkin exposed to H 2 O 2 (10 mM), followed by treatment with increasing concentrations of DTT (0-100 mM) prior to centrifugation and loading of the supernatant onto SDS-PAGE. (c,d) Circular dichroism spectra of soluble, untreated r-parkin at (c) T=0 and (d) soluble (black line) and aggregated (red line) states following incubation at 37°C for T=5 days. The protein secondary structure shifts from a predominant appearance of α-helix dominated state, as demonstrated by the positive band at 193 nm and negative bands 208 nm (black lines), to the appearance of β-pleated sheet formation, as demonstrated by negative bands at 218 nm and a rise in molar ellipticity with positive bands at 195 nm (red lines) during spontaneous oxidation. (e-f) Quantitative analyses of IAA-modified cysteines captured by LC-MS/MS for (e) untreated vs. H 2 O 2 -exposed r-parkin, and (f) soluble compared to insoluble (pellet) fractions. Each data point represents the log2-transformed total IAA-signal intensities of single cysteine residues (n=3 runs for each). The cysteine pool is shown with the mean ± SEM; significance **p

    Article Snippet: Protein staining methods All proteins were separated on pre-cast 4-12 % Bis-Tris SDS-PAGE gels (NPO321BOX, NPO322BOX, NPO336BOX) from Invitrogen using MES running buffer (50mM MES, 50mM Tris, 1mM EDTA and 0.1 % SDS, pH 7.3) and Laemmli loading buffer (10% SDS, 20% glycerol, 0.1% bromophenol blue, 0.125M Tris HCl, 200mM DTT or β-mercaptoethanol).

    Techniques: Silver Staining, Staining, Centrifugation, SDS Page, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Transformation Assay

    TCR-mediated signaling in TRIM-deficient CD4 + T cells. (A) TRIM expression in T cells. Postnuclear lysates were prepared from freshly isolated thymocytes, lymph node cells (LN), or purified CD4 + and CD8 + splenocytes, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with TRIM04 MAb. The blot was reprobed with anti-Erk1/2 to demonstrate equal loading. Numbers below the blot represent relative TRIM expression compared to the lowest. (B) Global tyrosine phosphorylation and MAP family kinase activation are normal, but Akt phosphorylation is enhanced in TRIM-deficient mice. Purified CD4 + splenic T cells were stimulated for the indicated periods of time with the CD3 MAb 145-2C11. The cells were lysed and processed for Western blot analysis. The blots were probed with anti-phosphospecific antibodies and then stripped and reprobed with anti-Erk1/2, anti-ZAP-70, and anti-Akt to show equal loading. Data are representative of five separate experiments. (C) Analysis of Ca 2+ mobilization. Purified splenic CD4 + cells were loaded with indo-1-AM and incubated with 10 μg/ml of biotinylated anti-CD3 (145-2C11). The arrows indicate the addition of streptavidin and ionomycin (Iono), respectively. (D) TRIM regulation of Akt phosphorylation depends on PI3K activity. Purified CD4 + splenic T cells were stimulated for 2 min with CD3 MAb alone or with CD3 plus CD28 MAbs in the presence or absence of the PI3K inhibitor wortmannin. The level of Akt activation was measured by using a phosphospecific anti-Akt antibody and the equal loading by using an anti-Akt antibody. stim., stimulation.

    Journal: Molecular and Cellular Biology

    Article Title: Normal T-Cell Development and Immune Functions in TRIM-Deficient Mice

    doi: 10.1128/MCB.26.9.3639-3648.2006

    Figure Lengend Snippet: TCR-mediated signaling in TRIM-deficient CD4 + T cells. (A) TRIM expression in T cells. Postnuclear lysates were prepared from freshly isolated thymocytes, lymph node cells (LN), or purified CD4 + and CD8 + splenocytes, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with TRIM04 MAb. The blot was reprobed with anti-Erk1/2 to demonstrate equal loading. Numbers below the blot represent relative TRIM expression compared to the lowest. (B) Global tyrosine phosphorylation and MAP family kinase activation are normal, but Akt phosphorylation is enhanced in TRIM-deficient mice. Purified CD4 + splenic T cells were stimulated for the indicated periods of time with the CD3 MAb 145-2C11. The cells were lysed and processed for Western blot analysis. The blots were probed with anti-phosphospecific antibodies and then stripped and reprobed with anti-Erk1/2, anti-ZAP-70, and anti-Akt to show equal loading. Data are representative of five separate experiments. (C) Analysis of Ca 2+ mobilization. Purified splenic CD4 + cells were loaded with indo-1-AM and incubated with 10 μg/ml of biotinylated anti-CD3 (145-2C11). The arrows indicate the addition of streptavidin and ionomycin (Iono), respectively. (D) TRIM regulation of Akt phosphorylation depends on PI3K activity. Purified CD4 + splenic T cells were stimulated for 2 min with CD3 MAb alone or with CD3 plus CD28 MAbs in the presence or absence of the PI3K inhibitor wortmannin. The level of Akt activation was measured by using a phosphospecific anti-Akt antibody and the equal loading by using an anti-Akt antibody. stim., stimulation.

    Article Snippet: Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride or nitrocellulose membranes, and blotted with the following antibodies: anti-phosphotyrosine (4G10), anti-ERK1/2 (pT202/pT204), anti-JNK (pT183/pY185), anti-p38 (pT180/pT182), anti-phospho-Akt (S473), anti-Akt, all from Cell Signaling, and anti-ZAP70 (clone Z24820; Transduction Laboratories), anti-ERK1/2 (Cell Signaling), or β-actin (Sigma).

    Techniques: Expressing, Isolation, Purification, Polyacrylamide Gel Electrophoresis, Activation Assay, Mouse Assay, Western Blot, Incubation, Activity Assay