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    Thermo Fisher bis tris gels
    SDS PAGE showing ADR1 constructs translated in PURExpress Δ-Ribosome kit supplemented with high-salt-washed ribosomes isolated from HDB140, HDB143 (uL23 Δloop), or HDB144 (uL24 Δloop) as indicated. Translations were run on 12% <t>Bis-Tris</t> gels with <t>MOPS</t> running buffer.
    Bis Tris Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 23988 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bis tris gel
    Western blots of human milk adiponectin. ( A ) In native <t>PAGE,</t> 13 μL (13) or 10.4 μL (10) of milk (M) or serum (S) (13 μL of a 1:60 dilution) was applied to each well of a native PAGE 3–12% <t>Bis-Tris</t>
    Bis Tris Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 20757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher nupage bis tris gels
    Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% <t>NuPAGE</t> <t>Bis-Tris</t> gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).
    Nupage Bis Tris Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bis tris protein gels
    The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by <t>Native-PAGE</t> (4–8% <t>Tris–acetate</t> gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation
    Bis Tris Protein Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2933 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bis tris nupage gels
    Glycosylation studies of 5-HT3C, -D and -Ea. A , immunoprecipitation of metabolically labeled proteins of HEK293 cells expressing Myc-/HA-tagged 5-HT3C, -D, and -Ea subunits. Immunoprecipitation was carried out with an anti-HA or an anti-Myc antibody, respectively. Proteins were separated on a 4–12% <t>Bis-Tris</t> <t>NuPAGE</t> gel (Invitrogen) followed by autoradiography. To check for glycosylation of the respective subunit, one batch was treated with tunicamycin, and the other was not. Immunoreactive bands of approximately 50–60 kDa (HA-5-HT3C), approximately 30 kDa (HA-5-HT3D), and approximately 60 kDa (Myc-5-HT3Ea) were detectable for untreated cells. After tunicamycin treatment, no effect was detectable for 5-HT3D, whereas the band sizes for 5-HT3C and 5-HT3Ea were reduced to approximately 40 kDa, respectively. B , Western blot of N -glycosylation knock-out constructs generated by site-directed mutagenesis, affecting either one of four predicted N -glycosylation sites (N31Q, N59Q, N67Q, or N175Q) in 5-HT3C and 5-HT3Ea or all of them ( N > Q *). HEK293 cells were transfected with 5-HT3C or -Ea constructs, and one batch of the wild-type ( wt ) subunits was treated with tunicamycin ( Tun. ). Proteins were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and blotted on polyvinylidene difluoride membranes. Subunit-specific antibodies were used for detection following the Odyssey Western Blot Analysis protocol (Li-Cor Biosciences). Immunoreactive bands of approximately 40–55 kDa were detectable with the upmost band (55 kDa) missing in the case of the single knock-outs. The N > Q* knockouts and the tunicamycin-treated cells showed only one band at approximately 40 kDa.
    Bis Tris Nupage Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage
    Two wt ClpB subunits ensure binding of ClpB hybrids to aggregates in a DnaK-dependent manner. Binding of ClpB heterohexamers made of wt and ( A ) ClpB TT , ( B ) ClpB ΔM , or ( C ) ClpB ΔN subunits to protein aggregates. G6PDH 50 aggregates were diluted to 1 μM in refolding buffer containing 3 mM ATP, DnaK (3.5 μM), DnaJ (0.7 μM), and 5 µM of the corresponding ClpB homo or heterohexamer. After an incubation of 10 min, samples were centrifuged to obtain the pellets containing aggregate-bound chaperones, which were analyzed by 4–12% <t>Bis-Tris</t> <t>NuPAGE</t> (upper panels) to estimate the amount of each ClpB homo and heterohexamer bound to the aggregate, using known amounts of each protein as standard (bottom panels). Data are shown as mean ± s.d. of three independent experiments (* P
    Nupage, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher bis tris gradient gels
    MSF and Nedd5 coimmunoprecipitate in a septin complex in interphase HeLa cells. Immunoprecipitations were performed as described in MATERIALS AND METHODS using anti-MSF (A and C) or anti-Nedd5 (B and C) antibodies and analyzed by Western blotting with the indicated antibodies (A and B) or by Coomassie blue staining (C). Samples of the starting material were also analyzed (Ly), and immunoprecipitations with nonimmune IgG provided a control. Western blots shown are representative of three independent experiments. Relative molecular weights and the position of the IgG heavy chain are indicated. In C, the identities of the protein bands were determined by Western blotting (A, B, and our unpublished results). hCDC10 was also identified by MALDI-TOF analysis of an in-gel trypsin digest of the protein band (see text). The <t>NuPAGE</t> 4–12% <t>Bis-Tris</t> gradient gels shown in C are representative of at least five independent experiments. Asterisks (*) denote the upper two MSF immunoreactive species.
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    92
    Bio-Rad bis tris gels
    Biochemical analysis of native huntingtin in human brain. A , lysates were prepared from postmortem control ( Ctl ) and homozygous HD patient cortex. Proteins were separated with BNP using NativePAGE TM <t>Novex</t> 3–12% <t>Bis-Tris</t> gels and transferred to
    Bis Tris Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1889 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad bis tris gel
    This scheme illustrates EPF and perpendicular CoI inserted into the <t>TNT.</t> a EPF: CoI was transferred onto TNT in a semi dry transfer. The transfer unit was assembled from the bottom in the following order: (1) anode of a semi dry blotter; (2) 1× TGB-wetted filter paper; (3) TNT surface upward; (4) 6% Bis <t>Tris</t> gel; (5) TGB-wetted filter to which CoI was spotted at the titanium facing area; (6) cathode. b Scheme of perpendicular CoI inserted into the TNT
    Bis Tris Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SDS PAGE showing ADR1 constructs translated in PURExpress Δ-Ribosome kit supplemented with high-salt-washed ribosomes isolated from HDB140, HDB143 (uL23 Δloop), or HDB144 (uL24 Δloop) as indicated. Translations were run on 12% Bis-Tris gels with MOPS running buffer.

    Journal: eLife

    Article Title: The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding

    doi: 10.7554/eLife.36326

    Figure Lengend Snippet: SDS PAGE showing ADR1 constructs translated in PURExpress Δ-Ribosome kit supplemented with high-salt-washed ribosomes isolated from HDB140, HDB143 (uL23 Δloop), or HDB144 (uL24 Δloop) as indicated. Translations were run on 12% Bis-Tris gels with MOPS running buffer.

    Article Snippet: The samples were resolved on 12% Bis-Tris gels (Thermo Scientific) in MOPS buffer for ADR1 and MES buffer for Spectrin and Titin.

    Techniques: SDS Page, Construct, Isolation

    Purification of Aβ aggregates from the brain of an aged TgCRND8 mouse. a The purity of the isolated TgCRND8 Aβ aggregates was analysed by SDS-PAGE followed by either silver staining (left) or immunoblotting with the anti-Aβ antibody 6E10. b The Aβ isoform composition of the purified TgCRND8 Aβ aggregates was determined by separation on a Bicine/Tris 8 M urea gel followed by silver staining. PK-resistant Aβ fibrils prepared from various recombinant Aβ isoforms were used as controls. c Ultrastructural analysis of purified TgCRND8 Aβ aggregates by electron microscopy. Scale bars: 500 nm (left image) or 100 nm (right image)

    Journal: Acta Neuropathologica Communications

    Article Title: Prion-like propagation of β-amyloid aggregates in the absence of APP overexpression

    doi: 10.1186/s40478-018-0529-x

    Figure Lengend Snippet: Purification of Aβ aggregates from the brain of an aged TgCRND8 mouse. a The purity of the isolated TgCRND8 Aβ aggregates was analysed by SDS-PAGE followed by either silver staining (left) or immunoblotting with the anti-Aβ antibody 6E10. b The Aβ isoform composition of the purified TgCRND8 Aβ aggregates was determined by separation on a Bicine/Tris 8 M urea gel followed by silver staining. PK-resistant Aβ fibrils prepared from various recombinant Aβ isoforms were used as controls. c Ultrastructural analysis of purified TgCRND8 Aβ aggregates by electron microscopy. Scale bars: 500 nm (left image) or 100 nm (right image)

    Article Snippet: Samples were subjected to SDS-PAGE using Bolt 4–12% Bis-Tris Plus gels (ThermoFisher).

    Techniques: Purification, Isolation, SDS Page, Silver Staining, Recombinant, Electron Microscopy

    Identification of BACE1 cleavage sites in human Navβ2 by MS analysis . Reaction mixtures from an in vitro BACE1 cleavage assay were resolved on a 12% BisTris gel and full-length peptide and cleavage products were detected by silver staining. Only the incubation of the β2-peptide with BACE1 generates a ~2 kDa cleavage product. GL-189, a BACE1 inhibitor, significantly decreased the amount of cleavage product (lane 3). B) Western blot analysis of an in vitro BACE1 cleavage assay samples using anti-biotin-HRP conjugate. C) Table containing the major cleavage products and their predicted sequences determined by Mass spectrometry from control and the BACE1 reaction sample. D) Sequence of the β2-peptide synthesized for the in vitro cleavage assay. A biotinylated tyrosine amino acid is added to the N-terminus of the peptide for detection by anti-biotin-HRP conjugate. One major (big arrow) and one minor (small arrow) BACE1 cleavage site were detected by MS analysis.

    Journal: Molecular Neurodegeneration

    Article Title: Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    doi: 10.1186/1750-1326-5-61

    Figure Lengend Snippet: Identification of BACE1 cleavage sites in human Navβ2 by MS analysis . Reaction mixtures from an in vitro BACE1 cleavage assay were resolved on a 12% BisTris gel and full-length peptide and cleavage products were detected by silver staining. Only the incubation of the β2-peptide with BACE1 generates a ~2 kDa cleavage product. GL-189, a BACE1 inhibitor, significantly decreased the amount of cleavage product (lane 3). B) Western blot analysis of an in vitro BACE1 cleavage assay samples using anti-biotin-HRP conjugate. C) Table containing the major cleavage products and their predicted sequences determined by Mass spectrometry from control and the BACE1 reaction sample. D) Sequence of the β2-peptide synthesized for the in vitro cleavage assay. A biotinylated tyrosine amino acid is added to the N-terminus of the peptide for detection by anti-biotin-HRP conjugate. One major (big arrow) and one minor (small arrow) BACE1 cleavage site were detected by MS analysis.

    Article Snippet: 20-50 mg of protein were resolved on 12% BisTris gels (Invitrogen).

    Techniques: Mass Spectrometry, In Vitro, Cleavage Assay, Silver Staining, Incubation, Western Blot, Sequencing, Synthesized

    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

    CENP ‐C cupin dimerisation is critical for CAL 1 binding SEC analysis of (A) His‐CENP‐C 1264–1411 F1324R and His‐SUMO‐CAL1 841–979, (B) His‐CENP‐C 1264–1411 and His‐SUMO‐CAL1 841–979 I900R/K907A/Y908A and (C) His‐CENP‐C 1264–1411 L1357E/M1407E and His‐SUMO‐CAL1 841–979 . Samples were analysed using a Superdex 75 in 20 mM Tris–HCl pH 8.0, 0.1 M NaCl and 2 mM DTT. Corresponding fractions shown by SDS–PAGE with Coomassie stain underneath. Representative IF images and quantification of tethering assays. U2OS cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C with CAL1‐V5 to assess interaction. CENP‐C mutants F1324R, L1357E/M1407E and CAL1 mutant I900R/K907A/Y908A were tested in each construct separately. Scale bar: 10 μm ( n = 4 experiments except F1324 where n = 3 experiments). Representative IF images and quantification of in vivo tethering assays. Drosophila Schneider S2 cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C and CAL1‐HA to assess interaction. Arrows point to the LacO site. Scale bar is 5 μm ( n = 3 experiment, except CAL1 1900R/K907A/Y908A where n = 2 experiments). Data information: In (D), data presented as mean ± SEM of 3 or 4 experiments, n ≥ 22 cells per experiment. P ‐values were calculated using an Mann–Whitney test. In (E), data presented as mean ± SEM of 3 experiments, except for CAL1 mutant I900R/K907A/Y908A which was 2 experiments. n ≥ 44 cells per experiment, P ‐values were calculated using Mann–Whitney test (**** P

    Journal: The EMBO Journal

    Article Title: Structural basis for centromere maintenance by Drosophila CENP‐A chaperone CAL1

    doi: 10.15252/embj.2019103234

    Figure Lengend Snippet: CENP ‐C cupin dimerisation is critical for CAL 1 binding SEC analysis of (A) His‐CENP‐C 1264–1411 F1324R and His‐SUMO‐CAL1 841–979, (B) His‐CENP‐C 1264–1411 and His‐SUMO‐CAL1 841–979 I900R/K907A/Y908A and (C) His‐CENP‐C 1264–1411 L1357E/M1407E and His‐SUMO‐CAL1 841–979 . Samples were analysed using a Superdex 75 in 20 mM Tris–HCl pH 8.0, 0.1 M NaCl and 2 mM DTT. Corresponding fractions shown by SDS–PAGE with Coomassie stain underneath. Representative IF images and quantification of tethering assays. U2OS cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C with CAL1‐V5 to assess interaction. CENP‐C mutants F1324R, L1357E/M1407E and CAL1 mutant I900R/K907A/Y908A were tested in each construct separately. Scale bar: 10 μm ( n = 4 experiments except F1324 where n = 3 experiments). Representative IF images and quantification of in vivo tethering assays. Drosophila Schneider S2 cells containing a LacO array were transfected with GFP‐LacI‐CENP‐C and CAL1‐HA to assess interaction. Arrows point to the LacO site. Scale bar is 5 μm ( n = 3 experiment, except CAL1 1900R/K907A/Y908A where n = 2 experiments). Data information: In (D), data presented as mean ± SEM of 3 or 4 experiments, n ≥ 22 cells per experiment. P ‐values were calculated using an Mann–Whitney test. In (E), data presented as mean ± SEM of 3 experiments, except for CAL1 mutant I900R/K907A/Y908A which was 2 experiments. n ≥ 44 cells per experiment, P ‐values were calculated using Mann–Whitney test (**** P

    Article Snippet: Beads were then washed four times with 1 ml of buffer, then twice with 1 ml of 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 35 mM imidazole and 2 mM βME and eluted by boiling in SDS–PAGE loading dye before being separated on a Bolt™ 4–12% Bis‐Tris Plus gel (Invitrogen) run at 180 V for 1 h in MES buffer.

    Techniques: Binding Assay, SDS Page, Staining, Transfection, Mutagenesis, Construct, In Vivo, MANN-WHITNEY

    CAL 1 binds CENP ‐C by directly interacting with the evolutionarily conserved Cupin domain Schematic representation of the structural features of CENP‐C. Filled boxes represent domains. SEC profile of His‐SUMO‐CAL1 841–979 (black) , His‐CENP‐C 1264–1411 (red) and His‐SUMO‐CAL1 841–979 mixed with molar excess of His‐CENP‐C 1264–1411 (blue) and corresponding SDS–PAGE analysis of the fractions. Samples were analysed using Superdex 75 increase 10/300 in 20 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM DTT. Crystal structure of CENP‐C cupin domain determined at 1.7 Å resolution. Overall structure of CENP‐C cupin domain dimer. Amino acid residues involved in dimerisation are highlighted in zoomed in panels. Residues mutated to disrupt dimerisation are circled. SEC‐MALS analysis of CENP‐C 1264–1411 (black) and His‐CENP‐C 1264–1411 L1357E/M1407E (blue). Absorption at 280 nm (mAU, left y ‐axis) and molecular mass (kDa, right y ‐axis) are plotted against elution volume (ml, x ‐axis). Measured MW and the calculated subunit stoichiometry based on the predicted MW of different subunit compositions. Samples were analysed using either a Superdex 75 or a Superdex 200 increase 10/300 in 50 mM HEPES pH 8.0, 100 mM or 300 mM NaCl and 1 mM TCEP.

    Journal: The EMBO Journal

    Article Title: Structural basis for centromere maintenance by Drosophila CENP‐A chaperone CAL1

    doi: 10.15252/embj.2019103234

    Figure Lengend Snippet: CAL 1 binds CENP ‐C by directly interacting with the evolutionarily conserved Cupin domain Schematic representation of the structural features of CENP‐C. Filled boxes represent domains. SEC profile of His‐SUMO‐CAL1 841–979 (black) , His‐CENP‐C 1264–1411 (red) and His‐SUMO‐CAL1 841–979 mixed with molar excess of His‐CENP‐C 1264–1411 (blue) and corresponding SDS–PAGE analysis of the fractions. Samples were analysed using Superdex 75 increase 10/300 in 20 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM DTT. Crystal structure of CENP‐C cupin domain determined at 1.7 Å resolution. Overall structure of CENP‐C cupin domain dimer. Amino acid residues involved in dimerisation are highlighted in zoomed in panels. Residues mutated to disrupt dimerisation are circled. SEC‐MALS analysis of CENP‐C 1264–1411 (black) and His‐CENP‐C 1264–1411 L1357E/M1407E (blue). Absorption at 280 nm (mAU, left y ‐axis) and molecular mass (kDa, right y ‐axis) are plotted against elution volume (ml, x ‐axis). Measured MW and the calculated subunit stoichiometry based on the predicted MW of different subunit compositions. Samples were analysed using either a Superdex 75 or a Superdex 200 increase 10/300 in 50 mM HEPES pH 8.0, 100 mM or 300 mM NaCl and 1 mM TCEP.

    Article Snippet: Beads were then washed four times with 1 ml of buffer, then twice with 1 ml of 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 35 mM imidazole and 2 mM βME and eluted by boiling in SDS–PAGE loading dye before being separated on a Bolt™ 4–12% Bis‐Tris Plus gel (Invitrogen) run at 180 V for 1 h in MES buffer.

    Techniques: SDS Page

    CAL 1 can associate with CENP ‐A/H4 heterodimer and CENP ‐C simultaneously (left panel) Normalised sedimentation coefficient distribution (c(s)) for CAL1 841–979 (CAL1, blue), CENP‐C 1264–1411 (CENP‐C, red) and their equimolar mix (complex, purple) all at 10 mg/ml, demonstrating a significant increase in s 20 , w 0 consistent with the formation of a 2:1 complex. (right panel) Typical sedimentation equilibrium data for CAL1 841–979 (CAL1, blue), CENP‐C 1264–1411 (CENP‐C, red) and their equimolar mix (complex, purple) all at 10 mg/ml, demonstrating a significant increase in mass, consistent with the formation of a 2:1 complex. The data were fit with a single species model yielding masses of 20,253, 29,216 and 49,539 g/mol. The values reported in Fig EV5 C are based on data acquired for a range of concentrations. SEC profile of His‐CENP‐C 1264–1411 (red), His‐CAL1 1–160‐LL‐841–979 /CENP‐A 101–225 /H4 (black) and His‐ His‐CAL1 1–160‐LL‐841–979 /CENP‐A 101–225 /H4 mixed with molar excess of His‐CENP‐C 1264–1411 (blue) and corresponding SDS–PAGE analysis of the fractions. Samples were analysed using Superdex 200 increase 10/300 in 20 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM DTT. Schematic model of CAL1‐mediated loading of CENP‐A/H4 and CENP‐C binding at centromeres.

    Journal: The EMBO Journal

    Article Title: Structural basis for centromere maintenance by Drosophila CENP‐A chaperone CAL1

    doi: 10.15252/embj.2019103234

    Figure Lengend Snippet: CAL 1 can associate with CENP ‐A/H4 heterodimer and CENP ‐C simultaneously (left panel) Normalised sedimentation coefficient distribution (c(s)) for CAL1 841–979 (CAL1, blue), CENP‐C 1264–1411 (CENP‐C, red) and their equimolar mix (complex, purple) all at 10 mg/ml, demonstrating a significant increase in s 20 , w 0 consistent with the formation of a 2:1 complex. (right panel) Typical sedimentation equilibrium data for CAL1 841–979 (CAL1, blue), CENP‐C 1264–1411 (CENP‐C, red) and their equimolar mix (complex, purple) all at 10 mg/ml, demonstrating a significant increase in mass, consistent with the formation of a 2:1 complex. The data were fit with a single species model yielding masses of 20,253, 29,216 and 49,539 g/mol. The values reported in Fig EV5 C are based on data acquired for a range of concentrations. SEC profile of His‐CENP‐C 1264–1411 (red), His‐CAL1 1–160‐LL‐841–979 /CENP‐A 101–225 /H4 (black) and His‐ His‐CAL1 1–160‐LL‐841–979 /CENP‐A 101–225 /H4 mixed with molar excess of His‐CENP‐C 1264–1411 (blue) and corresponding SDS–PAGE analysis of the fractions. Samples were analysed using Superdex 200 increase 10/300 in 20 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM DTT. Schematic model of CAL1‐mediated loading of CENP‐A/H4 and CENP‐C binding at centromeres.

    Article Snippet: Beads were then washed four times with 1 ml of buffer, then twice with 1 ml of 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 35 mM imidazole and 2 mM βME and eluted by boiling in SDS–PAGE loading dye before being separated on a Bolt™ 4–12% Bis‐Tris Plus gel (Invitrogen) run at 180 V for 1 h in MES buffer.

    Techniques: Sedimentation, SDS Page, Binding Assay

    Western blots of human milk adiponectin. ( A ) In native PAGE, 13 μL (13) or 10.4 μL (10) of milk (M) or serum (S) (13 μL of a 1:60 dilution) was applied to each well of a native PAGE 3–12% Bis-Tris

    Journal:

    Article Title: Human Milk Adiponectin Is Associated with Infant Growth in Two Independent Cohorts

    doi: 10.1089/bfm.2008.0137

    Figure Lengend Snippet: Western blots of human milk adiponectin. ( A ) In native PAGE, 13 μL (13) or 10.4 μL (10) of milk (M) or serum (S) (13 μL of a 1:60 dilution) was applied to each well of a native PAGE 3–12% Bis-Tris

    Article Snippet: For native polyacrylamide gele electrophoresis (PAGE), 13 μL of the aqueous milk preparation (e.g., 10.4 μL plus 2.6 μL of water) or 13 μL of a 1:60 dilution of serum was combined with 5 μL of 4× loading buffer and 2 μL of G250 and applied to each well of a native PAGE 3–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

    Techniques: Western Blot, Clear Native PAGE

    Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Journal: Plant Biotechnology Journal

    Article Title: Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

    doi: 10.1111/pbi.12572

    Figure Lengend Snippet: Immunodetection of Cr LPAAT 1 expression in C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA ‐tagged Cr LPAAT 1 using anti‐ HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS ‐ PAGE gel was stained with blue dye (ProSieve EX Safe Stain— LONZA ) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS ‐ PAGE gel, and expression of Cr LPAAT 1 was detected using anti‐ HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE : pLM 21‐Cr LPAAT 1‐ HA overexpressors; three transformants ( OE 1, OE 2 and OE 3) are shown.

    Article Snippet: Briefly, around 20 μg of protein was loaded onto a 12% Bis Tris SDS‐PAGE gel (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Immunodetection, Expressing, SDS Page, Staining

    Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).

    Article Snippet: The proteins were resolved on 10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA), transferred onto nitrocellulose membranes (Invitrogen), which were probed with a goat anti-p24gag antibody (dilution 1∶3000, provided by L. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeled antibody (dilution 1∶10,000; Calbiochem, EMD chemicals, Gibbstown, NJ) or with plasma (1∶200 dilution) from DNA vaccinated mice followed by anti-mouse IgG-HRP labeled (1∶10,000 dilution, GE Healthcare, Piscataway, NJ).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Labeling, Expressing

    Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 µg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts (1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24 gag antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 µg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts (1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24 gag antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

    Article Snippet: The proteins were resolved on 10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA), transferred onto nitrocellulose membranes (Invitrogen), which were probed with a goat anti-p24gag antibody (dilution 1∶3000, provided by L. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeled antibody (dilution 1∶10,000; Calbiochem, EMD chemicals, Gibbstown, NJ) or with plasma (1∶200 dilution) from DNA vaccinated mice followed by anti-mouse IgG-HRP labeled (1∶10,000 dilution, GE Healthcare, Piscataway, NJ).

    Techniques: Expressing, Transfection, Cell Culture, Plasmid Preparation, Western Blot

    Western blot analysis of recombinant C2 phage expressing the fragment of CcOX1. 10 11 phage particles diluted in loading buffer were resolved on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature and immunoblotted for detection with anti-pIII antibody. Wild-type M13 phage was used as a control. Migration of the molecular mass standards as well as pIII and pIII–CcOX1 fusion protein are indicated by arrowheads.

    Journal: PLoS ONE

    Article Title: Amyloid-? Peptide Binds to Cytochrome C Oxidase Subunit 1

    doi: 10.1371/journal.pone.0042344

    Figure Lengend Snippet: Western blot analysis of recombinant C2 phage expressing the fragment of CcOX1. 10 11 phage particles diluted in loading buffer were resolved on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature and immunoblotted for detection with anti-pIII antibody. Wild-type M13 phage was used as a control. Migration of the molecular mass standards as well as pIII and pIII–CcOX1 fusion protein are indicated by arrowheads.

    Article Snippet: 1011 phage particles diluted in 16 µl of loading buffer were boiled 5 minutes and separated on 4–12% NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 45 min at room temperature as recommended by manufacturer.

    Techniques: Western Blot, Recombinant, Expressing, Migration

    Characterization of GP1 protein purified by Ni column. A) Size exclusion chromatography elution profile. GP1 protein sample was loaded onto a Superdex 200 HR 10/30 column with PBS as a running buffer. B) SDS-PAGE analysis. GP1 samples were loaded onto a 4–12% Bis-Tris protein gel and stained with ProBlue. Lane 1, nonreducing conditions; Lane 2, protein markers; Lane 3, reducing conditions. C) Western blot analysis. 0.5 μg of purified GP1 was electrophoresed on a 4–12% SDS-polyacrylamide gel, transferred by electroblotting to a PVDF membrane, and visualized by immunostaining as described in the text. Under nonreducing conditions: Lane 1, protein markers; Lane 2, purified GP1. D) BIAcore binding sensorgram between GP1 and mouse anti-Zaire GP monoclonal antibody. The antibody was immobilized on a CM5 chip and GP1 served as an analyte. The concentrations of GP1 was 40, 5, 0.625 and 0.078 μg/ml respectively. E) Western blot analysis of GP1 deglycosylation. Under reducing conditions: Lane 1, protein markers; Lane 2, neuraminidase treated GP1; Lane 3, untreated GP1; Lane 4, PNGase F treated GP1. F) SDS-PAGE analysis of GP1 (Lane 2) and GPΔMuc (Lane 3) under reducing conditions. Lane 1, protein markers.

    Journal: Protein expression and purification

    Article Title: Overexpression of Ebola virus envelope GP1 protein

    doi: 10.1016/j.pep.2017.04.010

    Figure Lengend Snippet: Characterization of GP1 protein purified by Ni column. A) Size exclusion chromatography elution profile. GP1 protein sample was loaded onto a Superdex 200 HR 10/30 column with PBS as a running buffer. B) SDS-PAGE analysis. GP1 samples were loaded onto a 4–12% Bis-Tris protein gel and stained with ProBlue. Lane 1, nonreducing conditions; Lane 2, protein markers; Lane 3, reducing conditions. C) Western blot analysis. 0.5 μg of purified GP1 was electrophoresed on a 4–12% SDS-polyacrylamide gel, transferred by electroblotting to a PVDF membrane, and visualized by immunostaining as described in the text. Under nonreducing conditions: Lane 1, protein markers; Lane 2, purified GP1. D) BIAcore binding sensorgram between GP1 and mouse anti-Zaire GP monoclonal antibody. The antibody was immobilized on a CM5 chip and GP1 served as an analyte. The concentrations of GP1 was 40, 5, 0.625 and 0.078 μg/ml respectively. E) Western blot analysis of GP1 deglycosylation. Under reducing conditions: Lane 1, protein markers; Lane 2, neuraminidase treated GP1; Lane 3, untreated GP1; Lane 4, PNGase F treated GP1. F) SDS-PAGE analysis of GP1 (Lane 2) and GPΔMuc (Lane 3) under reducing conditions. Lane 1, protein markers.

    Article Snippet: SDS-PAGE was performed using NuPAGE™ Novex™ 4–12% Bis-Tris polyacrylamide gels (Invitrogen) under reducing or nonreducing conditions.

    Techniques: Purification, Size-exclusion Chromatography, SDS Page, Staining, Western Blot, Immunostaining, Binding Assay, Chromatin Immunoprecipitation

    Co-elution of MA with BAF in pull-down assays in the presence of DNA. Pull-down assays were performed on a Ni chelating sepharose column equilibrated with binding buffer. BAF was then applied in binding buffer and the column was extensively washed. Sonicated salmon sperm DNA (50 µl 0.02 (lane 3) or 0.06 µg/µl (lane 4)) was then applied and the washing step was repeated. 50 µl of 0.15 µg/µl MA was then applied in binding buffer and the washing step was repeated. Finally, BAF was eluted with 70 µl 1 M imidazole. Proteins were electrophoresed in a 4–12% Bis Tris NuPAGE gel (Invitrogen) and stained with Coomassie. Lane 5 shows MA alone as a mobility standard. In the presence of DNA some BAF remains trapped on the column during the elution step because it forms a cross-bridged network with DNA; it elutes with SDS (data not shown).

    Journal: PLoS ONE

    Article Title: No Interaction of Barrier-to-Autointegration Factor (BAF) with HIV-1 MA, Cone-Rod Homeobox (Crx) or MAN1-C in Absence of DNA

    doi: 10.1371/journal.pone.0025123

    Figure Lengend Snippet: Co-elution of MA with BAF in pull-down assays in the presence of DNA. Pull-down assays were performed on a Ni chelating sepharose column equilibrated with binding buffer. BAF was then applied in binding buffer and the column was extensively washed. Sonicated salmon sperm DNA (50 µl 0.02 (lane 3) or 0.06 µg/µl (lane 4)) was then applied and the washing step was repeated. 50 µl of 0.15 µg/µl MA was then applied in binding buffer and the washing step was repeated. Finally, BAF was eluted with 70 µl 1 M imidazole. Proteins were electrophoresed in a 4–12% Bis Tris NuPAGE gel (Invitrogen) and stained with Coomassie. Lane 5 shows MA alone as a mobility standard. In the presence of DNA some BAF remains trapped on the column during the elution step because it forms a cross-bridged network with DNA; it elutes with SDS (data not shown).

    Article Snippet: Proteins were electrophoresed in a 4–12% Bis Tris NuPAGE gel (Invitrogen) and stained with Coomassie.

    Techniques: Co-Elution Assay, Binding Assay, Sonication, Staining

    Recombinant VP1 S domains can interact with their RdRps in vitro . (A) SDS-PAGE analysis of purified RdRps and SD. E. coli purified GII.4 and MNV RdRps and VP1 SDs were resolved by a 4 to 12% NuPage Novex Bis-Tris gel (Invitrogen, Carlsbad, CA) and visualized by staining with Coomassie brilliant blue. (B) Differential scanning fluorimetry (DSF) profile of purified GII.4 and MNV RdRps in the presence of SDs. DSF was used to measure the stability of purified GII.4 RdRp in the presence of GII.4 or MNV SD. Each sample combination was tested in triplicate, and the results were duplicated in at least two independent assays. (C) Determination of thermal stability of GII.4 and MNV RdRps in the presence of their SDs by DSF. The differences between the T m s of RdRp alone and RdRp plus SD were calculated (Δ T m ). Each sample combination was tested in triplicate, and the results were duplicated in at least two independent assays. The data shown are the derivatives of the change in the fluorescence of the sample over time [-R′ (T)].

    Journal: Journal of Virology

    Article Title: Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

    doi: 10.1128/JVI.01208-12

    Figure Lengend Snippet: Recombinant VP1 S domains can interact with their RdRps in vitro . (A) SDS-PAGE analysis of purified RdRps and SD. E. coli purified GII.4 and MNV RdRps and VP1 SDs were resolved by a 4 to 12% NuPage Novex Bis-Tris gel (Invitrogen, Carlsbad, CA) and visualized by staining with Coomassie brilliant blue. (B) Differential scanning fluorimetry (DSF) profile of purified GII.4 and MNV RdRps in the presence of SDs. DSF was used to measure the stability of purified GII.4 RdRp in the presence of GII.4 or MNV SD. Each sample combination was tested in triplicate, and the results were duplicated in at least two independent assays. (C) Determination of thermal stability of GII.4 and MNV RdRps in the presence of their SDs by DSF. The differences between the T m s of RdRp alone and RdRp plus SD were calculated (Δ T m ). Each sample combination was tested in triplicate, and the results were duplicated in at least two independent assays. The data shown are the derivatives of the change in the fluorescence of the sample over time [-R′ (T)].

    Article Snippet: Samples were subsequently resolved by 4 to 12% NuPage Novex Bis-Tris gels using MOPS (morpholinepropanesulfonic acid)-SDS running buffer (Invitrogen, Carlsbad, CA), transferred to PVDF membranes, and detected by a Western blot analysis using the appropriate antibodies.

    Techniques: Recombinant, In Vitro, SDS Page, Purification, Staining, Fluorescence

    CPV purification and characterization. A. Sucrose gradient purification . VLPs preparation from infected cell culture lysates purified by sucrose gradient centrifugation (10–40%). Bands of CPV-VLPs that were derivatized with OG-488 are visible in the gradient just above the middle of the tube (left panel) and appear fluorescent green under a UV-light source (right panel). B. SDS-PAGE analyses . The purified VLPs were subjected to electrophoresis in 4–12% Bis-tris gel and stained with SimplyBlue (Invitrogen) to reveal the proteins (left panel). The Seeblue plus protein molecular weight standards in kDa (Invitrogen) are indicated on the side of the gel picture (lane 1). Lanes 2 and 3 contain protein from CPV-VLPs derivatized with OG-488 and CPV-VLPs respectively. Prior to staining, the gel (right panel) visualized with a UV-light source showed a fluorescent 62 kDa band in the lane of OG-488 derivatized CPV-VLPs (lane 2f) and lacked any fluorescent bands in the native CPV-VLPs (lane 3f).

    Journal: Journal of Nanobiotechnology

    Article Title: Canine parvovirus-like particles, a novel nanomaterial for tumor targeting

    doi: 10.1186/1477-3155-4-2

    Figure Lengend Snippet: CPV purification and characterization. A. Sucrose gradient purification . VLPs preparation from infected cell culture lysates purified by sucrose gradient centrifugation (10–40%). Bands of CPV-VLPs that were derivatized with OG-488 are visible in the gradient just above the middle of the tube (left panel) and appear fluorescent green under a UV-light source (right panel). B. SDS-PAGE analyses . The purified VLPs were subjected to electrophoresis in 4–12% Bis-tris gel and stained with SimplyBlue (Invitrogen) to reveal the proteins (left panel). The Seeblue plus protein molecular weight standards in kDa (Invitrogen) are indicated on the side of the gel picture (lane 1). Lanes 2 and 3 contain protein from CPV-VLPs derivatized with OG-488 and CPV-VLPs respectively. Prior to staining, the gel (right panel) visualized with a UV-light source showed a fluorescent 62 kDa band in the lane of OG-488 derivatized CPV-VLPs (lane 2f) and lacked any fluorescent bands in the native CPV-VLPs (lane 3f).

    Article Snippet: CPV-VLPs denatured in SDS-PAGE sample buffer were separated in a 4–12% bis-tris polyacrylamide gel (Invitrogen, Carlsbad, CA) by employing a 200 V constant current for 35 minutes.

    Techniques: Purification, Infection, Cell Culture, Gradient Centrifugation, SDS Page, Electrophoresis, Staining, Molecular Weight

    The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation

    Journal: Nature Communications

    Article Title: A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

    doi: 10.1038/s41467-018-03509-0

    Figure Lengend Snippet: The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation

    Article Snippet: SDS-PAGE and Native-PAGE Proteins were separated by SDS-PAGE using NuPAGE™ 4–12% Bis-Tris protein gels (Invitrogen), or separated by Native-PAGE using Novex™ 10–20% Tris-glycine or NuPAGE™ 3–8% Tris–acetate protein gels (Invitrogen), as indicated.

    Techniques: Incubation, Clear Native PAGE, Silver Staining, Western Blot, Activity Assay, Standard Deviation

    Phos-tag gel electrophoresis analysis of HBV Cp phosphorylation status. ( A ) 293T cells were transfected with the indicated plasmids and lysed with lysis buffer at 3 days post transfection. Intracellular capsids were determined by particle gel assay. HBV Cp in total cell lysates were resolved by electrophoresis in a NuPAGE 12% Bis-Tris Protein Gel and detected by a western blot assay. β-actin served as a loading control ((lower panel). The same samples were resolved by phos-tag gel electrophoresis and Cp was detected by Western blot assay with antibody HBc-170A. ( B ) The lysates of pCI-HBc and pCI-HBc-7A transfected 293T cells and total lysates of AML12HBVpolY63F cells as well as samples of sucrose gradient centrifugation fractions 3 to 10 described in Fig 4 were resolved by a 12.7% Phos-tag gel electrophoresis and proteins were transferred onto a PVDF membrane. HBV Cp was detected with antibody HBc-170A.

    Journal: PLoS Pathogens

    Article Title: Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids

    doi: 10.1371/journal.ppat.1008669

    Figure Lengend Snippet: Phos-tag gel electrophoresis analysis of HBV Cp phosphorylation status. ( A ) 293T cells were transfected with the indicated plasmids and lysed with lysis buffer at 3 days post transfection. Intracellular capsids were determined by particle gel assay. HBV Cp in total cell lysates were resolved by electrophoresis in a NuPAGE 12% Bis-Tris Protein Gel and detected by a western blot assay. β-actin served as a loading control ((lower panel). The same samples were resolved by phos-tag gel electrophoresis and Cp was detected by Western blot assay with antibody HBc-170A. ( B ) The lysates of pCI-HBc and pCI-HBc-7A transfected 293T cells and total lysates of AML12HBVpolY63F cells as well as samples of sucrose gradient centrifugation fractions 3 to 10 described in Fig 4 were resolved by a 12.7% Phos-tag gel electrophoresis and proteins were transferred onto a PVDF membrane. HBV Cp was detected with antibody HBc-170A.

    Article Snippet: The hyper- and hypo-phosphorylated Cp were resolved by electrophoresis in a NuPAGE 12% Bis-Tris Protein Gel (Invitrogen) and transferred onto PVDF membrane (Invitrogen).

    Techniques: Nucleic Acid Electrophoresis, Transfection, Lysis, Electrophoresis, Western Blot, Gradient Centrifugation

    Coagonist pMHC enhance human T-cell response to antigens by increasing phosphorylation of molecules involved in proximal TCR signaling pathway. Human E183 CTL were stimulated by T-REx CHO cells panel for 5 or 30 min, lysed and the lysate was separated on NuPAGE Bis-Tris Gel. The filter was blotted with antibodies against pCD3ζ ( a ), pZap70 ( b ), pLAT ( c ), pPLC-γ1 ( d ), p-Erk1/2 ( e ), and pSHP-1 ( f ). The data are representative of three independent experiments. Uncropped western blotting images are presented in Supplementary Fig. 8

    Journal: Nature Communications

    Article Title: Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling

    doi: 10.1038/s41467-018-05288-0

    Figure Lengend Snippet: Coagonist pMHC enhance human T-cell response to antigens by increasing phosphorylation of molecules involved in proximal TCR signaling pathway. Human E183 CTL were stimulated by T-REx CHO cells panel for 5 or 30 min, lysed and the lysate was separated on NuPAGE Bis-Tris Gel. The filter was blotted with antibodies against pCD3ζ ( a ), pZap70 ( b ), pLAT ( c ), pPLC-γ1 ( d ), p-Erk1/2 ( e ), and pSHP-1 ( f ). The data are representative of three independent experiments. Uncropped western blotting images are presented in Supplementary Fig. 8

    Article Snippet: The samples were loaded in a 4–12% Bis-Tris gradient gel (NuPAGE, Invitrogen) and transferred to a PVDF membrane (Immobilon- FL Transfer Membrane, Merck Millipore).

    Techniques: CTL Assay, Western Blot

    Effect of NaCl concentrations (0–2 M) on the DNA precipitation using poly(ethyleneimine) (PEI) 0.5% (w/v) in Tris–HCl buffer (25 mM, pH 8). a SDS–PAGE analysis. Concentrations of ( b ) DAMP4 var -pexiganan protein and c DNA remaining in the solution after PEI addition

    Journal: AMB Express

    Article Title: Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity

    doi: 10.1186/s13568-018-0541-3

    Figure Lengend Snippet: Effect of NaCl concentrations (0–2 M) on the DNA precipitation using poly(ethyleneimine) (PEI) 0.5% (w/v) in Tris–HCl buffer (25 mM, pH 8). a SDS–PAGE analysis. Concentrations of ( b ) DAMP4 var -pexiganan protein and c DNA remaining in the solution after PEI addition

    Article Snippet: Protein samples were qualitatively analyzed by sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% Bis–Tris Precast Gels (Life Technologies, Mulgrave, Australia) mounted in a Bio-Rad XCell 3 system (Bio-Rad, Hercules, CA) with an aqueous buffer solution of 2-( N -morpholino)ethanesulfonic acid.

    Techniques: SDS Page

    Glycosylation studies of 5-HT3C, -D and -Ea. A , immunoprecipitation of metabolically labeled proteins of HEK293 cells expressing Myc-/HA-tagged 5-HT3C, -D, and -Ea subunits. Immunoprecipitation was carried out with an anti-HA or an anti-Myc antibody, respectively. Proteins were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) followed by autoradiography. To check for glycosylation of the respective subunit, one batch was treated with tunicamycin, and the other was not. Immunoreactive bands of approximately 50–60 kDa (HA-5-HT3C), approximately 30 kDa (HA-5-HT3D), and approximately 60 kDa (Myc-5-HT3Ea) were detectable for untreated cells. After tunicamycin treatment, no effect was detectable for 5-HT3D, whereas the band sizes for 5-HT3C and 5-HT3Ea were reduced to approximately 40 kDa, respectively. B , Western blot of N -glycosylation knock-out constructs generated by site-directed mutagenesis, affecting either one of four predicted N -glycosylation sites (N31Q, N59Q, N67Q, or N175Q) in 5-HT3C and 5-HT3Ea or all of them ( N > Q *). HEK293 cells were transfected with 5-HT3C or -Ea constructs, and one batch of the wild-type ( wt ) subunits was treated with tunicamycin ( Tun. ). Proteins were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and blotted on polyvinylidene difluoride membranes. Subunit-specific antibodies were used for detection following the Odyssey Western Blot Analysis protocol (Li-Cor Biosciences). Immunoreactive bands of approximately 40–55 kDa were detectable with the upmost band (55 kDa) missing in the case of the single knock-outs. The N > Q* knockouts and the tunicamycin-treated cells showed only one band at approximately 40 kDa.

    Journal: The Journal of Biological Chemistry

    Article Title: RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits *

    doi: 10.1074/jbc.M110.122838

    Figure Lengend Snippet: Glycosylation studies of 5-HT3C, -D and -Ea. A , immunoprecipitation of metabolically labeled proteins of HEK293 cells expressing Myc-/HA-tagged 5-HT3C, -D, and -Ea subunits. Immunoprecipitation was carried out with an anti-HA or an anti-Myc antibody, respectively. Proteins were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) followed by autoradiography. To check for glycosylation of the respective subunit, one batch was treated with tunicamycin, and the other was not. Immunoreactive bands of approximately 50–60 kDa (HA-5-HT3C), approximately 30 kDa (HA-5-HT3D), and approximately 60 kDa (Myc-5-HT3Ea) were detectable for untreated cells. After tunicamycin treatment, no effect was detectable for 5-HT3D, whereas the band sizes for 5-HT3C and 5-HT3Ea were reduced to approximately 40 kDa, respectively. B , Western blot of N -glycosylation knock-out constructs generated by site-directed mutagenesis, affecting either one of four predicted N -glycosylation sites (N31Q, N59Q, N67Q, or N175Q) in 5-HT3C and 5-HT3Ea or all of them ( N > Q *). HEK293 cells were transfected with 5-HT3C or -Ea constructs, and one batch of the wild-type ( wt ) subunits was treated with tunicamycin ( Tun. ). Proteins were separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and blotted on polyvinylidene difluoride membranes. Subunit-specific antibodies were used for detection following the Odyssey Western Blot Analysis protocol (Li-Cor Biosciences). Immunoreactive bands of approximately 40–55 kDa were detectable with the upmost band (55 kDa) missing in the case of the single knock-outs. The N > Q* knockouts and the tunicamycin-treated cells showed only one band at approximately 40 kDa.

    Article Snippet: Total protein was determined using the BCA Protein Assay kit (Pierce), and 10 μg of protein were loaded on 4–12% Bis-Tris NuPAGE gels (Invitrogen).

    Techniques: Immunoprecipitation, Metabolic Labelling, Labeling, Expressing, Autoradiography, Western Blot, Knock-Out, Construct, Generated, Mutagenesis, Transfection

    Two wt ClpB subunits ensure binding of ClpB hybrids to aggregates in a DnaK-dependent manner. Binding of ClpB heterohexamers made of wt and ( A ) ClpB TT , ( B ) ClpB ΔM , or ( C ) ClpB ΔN subunits to protein aggregates. G6PDH 50 aggregates were diluted to 1 μM in refolding buffer containing 3 mM ATP, DnaK (3.5 μM), DnaJ (0.7 μM), and 5 µM of the corresponding ClpB homo or heterohexamer. After an incubation of 10 min, samples were centrifuged to obtain the pellets containing aggregate-bound chaperones, which were analyzed by 4–12% Bis-Tris NuPAGE (upper panels) to estimate the amount of each ClpB homo and heterohexamer bound to the aggregate, using known amounts of each protein as standard (bottom panels). Data are shown as mean ± s.d. of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate

    doi: 10.1038/s41598-018-24140-5

    Figure Lengend Snippet: Two wt ClpB subunits ensure binding of ClpB hybrids to aggregates in a DnaK-dependent manner. Binding of ClpB heterohexamers made of wt and ( A ) ClpB TT , ( B ) ClpB ΔM , or ( C ) ClpB ΔN subunits to protein aggregates. G6PDH 50 aggregates were diluted to 1 μM in refolding buffer containing 3 mM ATP, DnaK (3.5 μM), DnaJ (0.7 μM), and 5 µM of the corresponding ClpB homo or heterohexamer. After an incubation of 10 min, samples were centrifuged to obtain the pellets containing aggregate-bound chaperones, which were analyzed by 4–12% Bis-Tris NuPAGE (upper panels) to estimate the amount of each ClpB homo and heterohexamer bound to the aggregate, using known amounts of each protein as standard (bottom panels). Data are shown as mean ± s.d. of three independent experiments (* P

    Article Snippet: The resulting pellets and controls, containing known amounts of native proteins, were analyzed by SDS–PAGE (7.5%) or NuPAGE (4–12% Bis-Tris) (Novex, Life Technologies), as indicated.

    Techniques: Binding Assay, Incubation

    MSF and Nedd5 coimmunoprecipitate in a septin complex in interphase HeLa cells. Immunoprecipitations were performed as described in MATERIALS AND METHODS using anti-MSF (A and C) or anti-Nedd5 (B and C) antibodies and analyzed by Western blotting with the indicated antibodies (A and B) or by Coomassie blue staining (C). Samples of the starting material were also analyzed (Ly), and immunoprecipitations with nonimmune IgG provided a control. Western blots shown are representative of three independent experiments. Relative molecular weights and the position of the IgG heavy chain are indicated. In C, the identities of the protein bands were determined by Western blotting (A, B, and our unpublished results). hCDC10 was also identified by MALDI-TOF analysis of an in-gel trypsin digest of the protein band (see text). The NuPAGE 4–12% Bis-Tris gradient gels shown in C are representative of at least five independent experiments. Asterisks (*) denote the upper two MSF immunoreactive species.

    Journal: Molecular Biology of the Cell

    Article Title: The Mammalian Septin MSF Localizes with Microtubules and Is Required for Completion of Cytokinesis

    doi: 10.1091/mbc.E02-01-0042

    Figure Lengend Snippet: MSF and Nedd5 coimmunoprecipitate in a septin complex in interphase HeLa cells. Immunoprecipitations were performed as described in MATERIALS AND METHODS using anti-MSF (A and C) or anti-Nedd5 (B and C) antibodies and analyzed by Western blotting with the indicated antibodies (A and B) or by Coomassie blue staining (C). Samples of the starting material were also analyzed (Ly), and immunoprecipitations with nonimmune IgG provided a control. Western blots shown are representative of three independent experiments. Relative molecular weights and the position of the IgG heavy chain are indicated. In C, the identities of the protein bands were determined by Western blotting (A, B, and our unpublished results). hCDC10 was also identified by MALDI-TOF analysis of an in-gel trypsin digest of the protein band (see text). The NuPAGE 4–12% Bis-Tris gradient gels shown in C are representative of at least five independent experiments. Asterisks (*) denote the upper two MSF immunoreactive species.

    Article Snippet: Supernatants were subjected to electrophoresis on NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen) according to the manufacturer's instructions and subsequently stained in Coomassie blue for 1 h, followed by destaining in 50% H2 O, 40% methanol, 10% acetic acid.

    Techniques: Western Blot, Staining

    Platelets stored without citrate buffer show Talin degradation upon thawing. A. Coomassie stained 4–12% Bis-Tris SDS-PAGE gel shows selective degradation of Talin in lysates from platelet pellets stored in the absence of citrate buffer (Lane 3) Vs no degradation in the presence of citrate (Lane 2). B. Immuno-blotting with anti-Talin antibody against the Talin N-terminal rod region (antibody 8D4, [27] ) shows that Talin is degraded to smaller fragments in three independent platelet samples stored in the absence of citrate buffer (Lanes 4–6) as compared to three independent platelet samples stored in the presence of citrate (lanes 1–3). Notice the slight reduction in the MW of full-length Talin band in lanes 4–6 as well as the presence of ∼37 kDa fragments in lanes 4–6. C. Similarly, immuno-blotting with anti-Talin antibody against the Talin C-terminal region (antibody C20) also shows that Talin is degraded to a smaller MW species in two independent platelet samples stored in the absence of citrate buffer (Lanes 3–4) as compared to two independent platelet samples stored in the presence of citrate (lanes 1–2). Protein MW markers are as labeled. D. Immuno-blotting with the anti-integrin β3 mAb shows no degradation of integrin β3 upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled. E. Immuno-blotting with the anti-β-actin mAb shows no β-actin degradation upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled.

    Journal: PLoS ONE

    Article Title: Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

    doi: 10.1371/journal.pone.0007627

    Figure Lengend Snippet: Platelets stored without citrate buffer show Talin degradation upon thawing. A. Coomassie stained 4–12% Bis-Tris SDS-PAGE gel shows selective degradation of Talin in lysates from platelet pellets stored in the absence of citrate buffer (Lane 3) Vs no degradation in the presence of citrate (Lane 2). B. Immuno-blotting with anti-Talin antibody against the Talin N-terminal rod region (antibody 8D4, [27] ) shows that Talin is degraded to smaller fragments in three independent platelet samples stored in the absence of citrate buffer (Lanes 4–6) as compared to three independent platelet samples stored in the presence of citrate (lanes 1–3). Notice the slight reduction in the MW of full-length Talin band in lanes 4–6 as well as the presence of ∼37 kDa fragments in lanes 4–6. C. Similarly, immuno-blotting with anti-Talin antibody against the Talin C-terminal region (antibody C20) also shows that Talin is degraded to a smaller MW species in two independent platelet samples stored in the absence of citrate buffer (Lanes 3–4) as compared to two independent platelet samples stored in the presence of citrate (lanes 1–2). Protein MW markers are as labeled. D. Immuno-blotting with the anti-integrin β3 mAb shows no degradation of integrin β3 upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled. E. Immuno-blotting with the anti-β-actin mAb shows no β-actin degradation upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled.

    Article Snippet: Western blot analyses Platelet samples were separated by SDS-PAGE using a 4–12% gradient Bis-Tris gels (Invitrogen, CA USA) under reducing conditions and electroblotted onto PVDF membranes (Bio-Rad Laboratories, CA).

    Techniques: Staining, SDS Page, Labeling, Western Blot

    1D SDS-PAGE analysis of purified platelet membrane fraction. Platelet membrane was prepared as described in text. All fractions were analyzed using a 4–12% Tris-acetate pre-cast SDS-PAGE gel. Image of the coomassie stained gel shows that membrane fraction is highly enriched for membrane-associated proteins, such as integrin αIIb and β3 chains (arrows). Lane 1 = Molecular Weight markers (SeeBlue Plus2 from Invitrogen), Lane 2 = total platelet lysate, Lane 3 = cytoplasmic fraction after fractionation of platelet lysate on a 40% sucrose gradient, Lane 4 = extracted membrane fraction, Lane 5 = insoluble pellet after membrane fraction extraction.

    Journal: PLoS ONE

    Article Title: Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

    doi: 10.1371/journal.pone.0007627

    Figure Lengend Snippet: 1D SDS-PAGE analysis of purified platelet membrane fraction. Platelet membrane was prepared as described in text. All fractions were analyzed using a 4–12% Tris-acetate pre-cast SDS-PAGE gel. Image of the coomassie stained gel shows that membrane fraction is highly enriched for membrane-associated proteins, such as integrin αIIb and β3 chains (arrows). Lane 1 = Molecular Weight markers (SeeBlue Plus2 from Invitrogen), Lane 2 = total platelet lysate, Lane 3 = cytoplasmic fraction after fractionation of platelet lysate on a 40% sucrose gradient, Lane 4 = extracted membrane fraction, Lane 5 = insoluble pellet after membrane fraction extraction.

    Article Snippet: Western blot analyses Platelet samples were separated by SDS-PAGE using a 4–12% gradient Bis-Tris gels (Invitrogen, CA USA) under reducing conditions and electroblotted onto PVDF membranes (Bio-Rad Laboratories, CA).

    Techniques: SDS Page, Purification, Staining, Molecular Weight, Fractionation

    Talin is protectected from degradation during platelet thawing/lysis when stored in the presence of citrate. A. SDS-PAGE analysis of ten independent isolated platelet samples. Platelet lysates from five individual platelet samples isolated from fresh whole blood from healthy subjects (lanes 1–5) and five individual platelets samples obtained from the platelet rich plasma (PRP) (from blood bank) (lanes 6–10) were analyzed using 4–12% Bis-Tris SDS-PAGE. Images of the coomassie stained gels show that all ten samples have a very similar protein expression pattern and no noticeable Talin degradation (when compared with lane 3, Figure 3A ). Lane M = Protein MW markers. B–C. Western Blots show no Talin degradation in the presence of citrate in ten platelet samples. Platelet lysates from five individual platelet samples isolated from fresh whole blood (B) and five individual samples from platelets isolated from PRP (C) were analyzed by 4–12% Bis-Tris SDS-PAGE followed by immuno-blotting with anti-Talin antibody C-20. All ten samples show a single band for Talin with minimal degradation. Protein MW markers are as labeled.

    Journal: PLoS ONE

    Article Title: Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

    doi: 10.1371/journal.pone.0007627

    Figure Lengend Snippet: Talin is protectected from degradation during platelet thawing/lysis when stored in the presence of citrate. A. SDS-PAGE analysis of ten independent isolated platelet samples. Platelet lysates from five individual platelet samples isolated from fresh whole blood from healthy subjects (lanes 1–5) and five individual platelets samples obtained from the platelet rich plasma (PRP) (from blood bank) (lanes 6–10) were analyzed using 4–12% Bis-Tris SDS-PAGE. Images of the coomassie stained gels show that all ten samples have a very similar protein expression pattern and no noticeable Talin degradation (when compared with lane 3, Figure 3A ). Lane M = Protein MW markers. B–C. Western Blots show no Talin degradation in the presence of citrate in ten platelet samples. Platelet lysates from five individual platelet samples isolated from fresh whole blood (B) and five individual samples from platelets isolated from PRP (C) were analyzed by 4–12% Bis-Tris SDS-PAGE followed by immuno-blotting with anti-Talin antibody C-20. All ten samples show a single band for Talin with minimal degradation. Protein MW markers are as labeled.

    Article Snippet: Western blot analyses Platelet samples were separated by SDS-PAGE using a 4–12% gradient Bis-Tris gels (Invitrogen, CA USA) under reducing conditions and electroblotted onto PVDF membranes (Bio-Rad Laboratories, CA).

    Techniques: Lysis, SDS Page, Isolation, Staining, Expressing, Western Blot, Labeling

    Biochemical analysis of native huntingtin in human brain. A , lysates were prepared from postmortem control ( Ctl ) and homozygous HD patient cortex. Proteins were separated with BNP using NativePAGE TM Novex 3–12% Bis-Tris gels and transferred to

    Journal: The Journal of Biological Chemistry

    Article Title: Native Mutant Huntingtin in Human Brain: EVIDENCE FOR PREVALENCE OF FULL-LENGTH MONOMER*

    doi: 10.1074/jbc.M111.286609

    Figure Lengend Snippet: Biochemical analysis of native huntingtin in human brain. A , lysates were prepared from postmortem control ( Ctl ) and homozygous HD patient cortex. Proteins were separated with BNP using NativePAGE TM Novex 3–12% Bis-Tris gels and transferred to

    Article Snippet: Proteins were separated in NativePAGETM Novex 3–12 or 4–16% Bis-Tris gels and transferred to PVDF at 100 V for 1 h using the Mini Trans-Blot Cell (Bio-Rad).

    Techniques: CTL Assay

    This scheme illustrates EPF and perpendicular CoI inserted into the TNT. a EPF: CoI was transferred onto TNT in a semi dry transfer. The transfer unit was assembled from the bottom in the following order: (1) anode of a semi dry blotter; (2) 1× TGB-wetted filter paper; (3) TNT surface upward; (4) 6% Bis Tris gel; (5) TGB-wetted filter to which CoI was spotted at the titanium facing area; (6) cathode. b Scheme of perpendicular CoI inserted into the TNT

    Journal: Journal of Nanobiotechnology

    Article Title: Establishment of perpendicular protrusion of type I collagen on TiO2 nanotube surface as a priming site of peri-implant connective fibers

    doi: 10.1186/s12951-019-0467-1

    Figure Lengend Snippet: This scheme illustrates EPF and perpendicular CoI inserted into the TNT. a EPF: CoI was transferred onto TNT in a semi dry transfer. The transfer unit was assembled from the bottom in the following order: (1) anode of a semi dry blotter; (2) 1× TGB-wetted filter paper; (3) TNT surface upward; (4) 6% Bis Tris gel; (5) TGB-wetted filter to which CoI was spotted at the titanium facing area; (6) cathode. b Scheme of perpendicular CoI inserted into the TNT

    Article Snippet: The transfer unit was assembled in the following order from the bottom: (1) anode of a semi dry blotter (Trans-Blot Turbo Transfer System® , BIO-RAD Laboratory, CA, USA); (2) 1× tris glycine buffer (TGB)-wetted filter paper; (3) TiO2 or TNT surface upward of titanium; (4) 6% Bis Tris gel (BIO RAD Laboratory); (5) CoI-spotted TGB-wetted filter; and (6) cathode.

    Techniques: