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  • 99
    Thermo Fisher nupage
    Decreased cellular uptake of BoNT/A complex is not due to the proteolytic degradation of holotoxin nor binding of toxin to probiotics. BoNT/A was added to either Escherichia coli MG1655, or probiotics and incubated for 4 h at 37 °C. Soluble supernatant (S) and insoluble pellet (P) fractions were precipitated with trichloracetic acid (TCA). Precipitates were solubilized with sample loading buffer and loaded onto 10% <t>Bis-Tris</t> <t>NuPage</t> gels. Gels were transferred onto PVDF membranes and incubated with primary polyclonal antibody to BoNT/A (Metabiologics) and secondary goat anti-rabbit-HRP. Western blot was developed using Pierce SuperSignal ECL substrate. ( A ) Mean percent signal of BoNT/A in each fraction was quantified from four independent experiments ± SEM using FluorChem SP (Alpha Innotech); ( B ) Representative Western depicting the presence of full length BoNT/A. Statistical significance was determined by a two-tailed unpaired Student’s t -test, (*) p
    Nupage, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage lds
    SDS-PAGE of snake ( Agkistrodon piscivorus leucostoma ) total venom under denaturing but non-reducing condition <t>(NuPAGE</t> <t>LDS</t> sample buffer). Sample of 30 ul total venom was subjected to a 4–12% NuPAGE Novex Bis-Tris mini gel with MES running buffer.
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    99
    Thermo Fisher bis tris gels
    <t>SDS-PAGE</t> of purified S. mutans NADH oxidase 2 . Standard: SeeBlue ® Plus2 Prestained Protein Standard; lane 1: wild-type; lane 2: 193R194H, SDS-PAGE was performed with an Invitrogen NuPAGE ® system with a 4-12% <t>Bis-Tris</t> Gel and Coomassie staining
    Bis Tris Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24026 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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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 2741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage transfer buffer
    ApoD and LCAT proteins, but not ApoA1 protein, were present in keratocytes. In a Western blot assay, lysates from keratocytes, dermal fibroblasts and HepG2 cells were solubilized and loaded onto 4 to 12% <t>NuPAGE</t> <t>Bis-Tris</t> gradient gels. After electrophoresis, resolved proteins were transferred onto a nitrocellulose membrane and incubated with antibodies against either ApoA1 ( a ), ApoD ( c ) or LCAT ( e ). Anti-β-Actin served as a loading control ( b , d , f ).
    Nupage Transfer Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xcell ii blot module
    ApoD and LCAT proteins, but not ApoA1 protein, were present in keratocytes. In a Western blot assay, lysates from keratocytes, dermal fibroblasts and HepG2 cells were solubilized and loaded onto 4 to 12% <t>NuPAGE</t> <t>Bis-Tris</t> gradient gels. After electrophoresis, resolved proteins were transferred onto a nitrocellulose membrane and incubated with antibodies against either ApoA1 ( a ), ApoD ( c ) or LCAT ( e ). Anti-β-Actin served as a loading control ( b , d , f ).
    Xcell Ii Blot Module, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage mes sds running buffer
    Silver-stained <t>SDS-PAGE</t> gel (20% gradient <t>NuPAGE</t> system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ].
    Nupage Mes Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage sample reducing agent
    Silver-stained <t>SDS-PAGE</t> gel (20% gradient <t>NuPAGE</t> system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ). A pre-stained ladder was loaded (6 μL), and 23 μL of each sample was loaded into each lane. The predicted molecular weight of RodA is 15.2 kDa. The identity of these bands as RodA at these locations was confirmed by mass spectrometry in [ 38 ]. These are likely glycoforms of the protein [ 57 ].
    Nupage Sample Reducing Agent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage mops sds running buffer
    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% <t>SDS-PAGE),</t> using NuPAGE Bis-Tris gels with <t>MOPS-SDS</t> Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p
    Nupage Mops Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xcell surelock mini cell
    A) SDS reduced 10–20% Tricine gel of mojastin disintegrins. The gel was run under reducing condition with 1X Tricine SDS running buffer using an <t>XCell</t> <t>SureLock</t> Mini cell at 125V for 90 min. The gel was stained with Simply Blue Safe Stain for 1 h and destained overnight with Milli-Q water. Lane 1: SeeBlue Plus2 markers; Lane 2: native mojastin; Lane 3: r-mojastin 1; and Lane 4: r-mojastin 1-GST. B) MALDI-TOF mass spectrums of mojastin disintegrins. The samples were run in a linear mode using an ion source 1 of 20.00 kV, ion source 2 of 18.40 kV, a lens of 9.00 kV, and a pulse ion extraction of 350 ns on a Bruker Daltonics MALDI-TOF-TOF. B) r-mojastin 1; C) native mojastin; and D) r-mojastin 1-GST.
    Xcell Surelock Mini Cell, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Decreased cellular uptake of BoNT/A complex is not due to the proteolytic degradation of holotoxin nor binding of toxin to probiotics. BoNT/A was added to either Escherichia coli MG1655, or probiotics and incubated for 4 h at 37 °C. Soluble supernatant (S) and insoluble pellet (P) fractions were precipitated with trichloracetic acid (TCA). Precipitates were solubilized with sample loading buffer and loaded onto 10% Bis-Tris NuPage gels. Gels were transferred onto PVDF membranes and incubated with primary polyclonal antibody to BoNT/A (Metabiologics) and secondary goat anti-rabbit-HRP. Western blot was developed using Pierce SuperSignal ECL substrate. ( A ) Mean percent signal of BoNT/A in each fraction was quantified from four independent experiments ± SEM using FluorChem SP (Alpha Innotech); ( B ) Representative Western depicting the presence of full length BoNT/A. Statistical significance was determined by a two-tailed unpaired Student’s t -test, (*) p

    Journal: Toxins

    Article Title: Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A

    doi: 10.3390/toxins8120377

    Figure Lengend Snippet: Decreased cellular uptake of BoNT/A complex is not due to the proteolytic degradation of holotoxin nor binding of toxin to probiotics. BoNT/A was added to either Escherichia coli MG1655, or probiotics and incubated for 4 h at 37 °C. Soluble supernatant (S) and insoluble pellet (P) fractions were precipitated with trichloracetic acid (TCA). Precipitates were solubilized with sample loading buffer and loaded onto 10% Bis-Tris NuPage gels. Gels were transferred onto PVDF membranes and incubated with primary polyclonal antibody to BoNT/A (Metabiologics) and secondary goat anti-rabbit-HRP. Western blot was developed using Pierce SuperSignal ECL substrate. ( A ) Mean percent signal of BoNT/A in each fraction was quantified from four independent experiments ± SEM using FluorChem SP (Alpha Innotech); ( B ) Representative Western depicting the presence of full length BoNT/A. Statistical significance was determined by a two-tailed unpaired Student’s t -test, (*) p

    Article Snippet: Protein samples were TCA precipitated and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with NuPAGE 10% Bis-Tris gels (Invitrogen) followed by Western blotting.

    Techniques: Binding Assay, Incubation, Western Blot, Two Tailed Test

    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

    SDS-PAGE analysis of viral antigen composition of CVA16 and EV71 viral particles. CVA16 P-particles (lane 1), CVA16 R-particles (lane 2), EV71 P-particles (lane 3), and EV71 R-particles (lane 4) were analyzed on a NuPAGE 4–12% Birs-Tris Gel.

    Journal: PLoS ONE

    Article Title: Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

    doi: 10.1371/journal.pone.0049973

    Figure Lengend Snippet: SDS-PAGE analysis of viral antigen composition of CVA16 and EV71 viral particles. CVA16 P-particles (lane 1), CVA16 R-particles (lane 2), EV71 P-particles (lane 3), and EV71 R-particles (lane 4) were analyzed on a NuPAGE 4–12% Birs-Tris Gel.

    Article Snippet: SDS-PAGE analysis and Western blotting SDS-PAGE analysis of purified CVA16 virus fractions was performed in a NuPAGE 4–12% Bis-Tris Gel (Invitrogen, CA USA) according to the protocol suggested by the manufacturer.

    Techniques: SDS Page

    Western blot analysis of reactivity of enterovirus antigens with mouse anti-sera raised against formalin-inactivated CVA16 particles. R-particles derived from CVA16 and EV71 were separated on a NuPAGE 4–12% Bis-Tris Gel and analyzed using different antibodies: ( A ) anti-sera generated from formalin-inactivated CVA16 P-particles; ( B ) anti-sera generated from formalin-inactivated CVA16 R-particles; and ( C ) EV71-specific monoclonal antibody MAb N1.

    Journal: PLoS ONE

    Article Title: Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

    doi: 10.1371/journal.pone.0049973

    Figure Lengend Snippet: Western blot analysis of reactivity of enterovirus antigens with mouse anti-sera raised against formalin-inactivated CVA16 particles. R-particles derived from CVA16 and EV71 were separated on a NuPAGE 4–12% Bis-Tris Gel and analyzed using different antibodies: ( A ) anti-sera generated from formalin-inactivated CVA16 P-particles; ( B ) anti-sera generated from formalin-inactivated CVA16 R-particles; and ( C ) EV71-specific monoclonal antibody MAb N1.

    Article Snippet: SDS-PAGE analysis and Western blotting SDS-PAGE analysis of purified CVA16 virus fractions was performed in a NuPAGE 4–12% Bis-Tris Gel (Invitrogen, CA USA) according to the protocol suggested by the manufacturer.

    Techniques: Western Blot, Derivative Assay, Generated

    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

    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

    MassSpec analyses of selected polyprotein cleavage products. (A) nsp1α. (B) nsp1α+β. (C) nsp1α+β+γ. Products from in vitro transcription/translation reactions were separated on a 12% NuPAGE bis-tris gel

    Journal: Journal of Virology

    Article Title: Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases

    doi: 10.1128/JVI.01020-14

    Figure Lengend Snippet: MassSpec analyses of selected polyprotein cleavage products. (A) nsp1α. (B) nsp1α+β. (C) nsp1α+β+γ. Products from in vitro transcription/translation reactions were separated on a 12% NuPAGE bis-tris gel

    Article Snippet: The precipitated peptides were separated on different lanes of a 12% NuPAGE bis-tris (2-[bisamino]-2–1,3-propanediol) gel using NuPAGE MOPS (morpholinepropanesulfonic acid) SDS buffer (Life Technology).

    Techniques: In Vitro

    SDS-PAGE of snake ( Agkistrodon piscivorus leucostoma ) total venom under denaturing but non-reducing condition (NuPAGE LDS sample buffer). Sample of 30 ul total venom was subjected to a 4–12% NuPAGE Novex Bis-Tris mini gel with MES running buffer.

    Journal:

    Article Title: Complementary DNA sequencing and identification of mRNAs from venomous gland of Agkistrodon piscivorus leucostoma

    doi: 10.1016/j.toxicon.2008.03.028

    Figure Lengend Snippet: SDS-PAGE of snake ( Agkistrodon piscivorus leucostoma ) total venom under denaturing but non-reducing condition (NuPAGE LDS sample buffer). Sample of 30 ul total venom was subjected to a 4–12% NuPAGE Novex Bis-Tris mini gel with MES running buffer.

    Article Snippet: Thirty micrograms of crude venom from A. p. leucostoma was heated in NuPAGE LDS sample buffer (Invitrogen) containing SDS without reducing agents for 10 min and subjected to a NU-PAGE 4–12% Bis-Tris gel (1 mm thick) using NuPAGE MES SDS Running buffer (Invitrogen).

    Techniques: SDS Page

    Direct interaction between 4ICD and STAT5A is mediated by HER4 Y984. HEK 293T cells were transfected with the indicated expression vectors and cell lysates were prepared at 48 h. post-transfection in EBC buffer. For immunoprecipitations, 500 μg of cleared cell lysates were incubated with HER4 or STAT5A specific antibodies at 4 °C overnight. Immune complexes were recovered by adding Protein A sepharose to each immunoprecipitation reaction and incubating for 3 h at 4 °C, and finally eluted into 60 μl of NuPAGE LDS Sample Buffer with Reducing Agent. Twenty μl of eluted immunoprecipitation reactions or 20 μg of EBC lysate (Input) was probed by western blot using the indicated immunoblot (IB) antibodies.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Direct coupling of the HER4 intracellular domain (4ICD) and STAT5A signaling is required to induce mammary epithelial cell differentiation

    doi: 10.1016/j.bbrep.2016.07.015

    Figure Lengend Snippet: Direct interaction between 4ICD and STAT5A is mediated by HER4 Y984. HEK 293T cells were transfected with the indicated expression vectors and cell lysates were prepared at 48 h. post-transfection in EBC buffer. For immunoprecipitations, 500 μg of cleared cell lysates were incubated with HER4 or STAT5A specific antibodies at 4 °C overnight. Immune complexes were recovered by adding Protein A sepharose to each immunoprecipitation reaction and incubating for 3 h at 4 °C, and finally eluted into 60 μl of NuPAGE LDS Sample Buffer with Reducing Agent. Twenty μl of eluted immunoprecipitation reactions or 20 μg of EBC lysate (Input) was probed by western blot using the indicated immunoblot (IB) antibodies.

    Article Snippet: Immune complexes were collected by incubating with Protein A sepharose (Roche) at 4 °C for 3 h. and eluted by boiling in 60 μl of NuPAGE LDS Sample Buffer (4X) (Life Technologies) containing NuPAGE Sample Reducing Agent (10X) (Life Technologies).

    Techniques: Transfection, Expressing, Incubation, Immunoprecipitation, Western Blot

    SDS-PAGE of purified S. mutans NADH oxidase 2 . Standard: SeeBlue ® Plus2 Prestained Protein Standard; lane 1: wild-type; lane 2: 193R194H, SDS-PAGE was performed with an Invitrogen NuPAGE ® system with a 4-12% Bis-Tris Gel and Coomassie staining

    Journal: Computational and Structural Biotechnology Journal

    Article Title: Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations

    doi: 10.5936/csbj.201402005

    Figure Lengend Snippet: SDS-PAGE of purified S. mutans NADH oxidase 2 . Standard: SeeBlue ® Plus2 Prestained Protein Standard; lane 1: wild-type; lane 2: 193R194H, SDS-PAGE was performed with an Invitrogen NuPAGE ® system with a 4-12% Bis-Tris Gel and Coomassie staining

    Article Snippet: For SDS-PAGE NuPAGE® 4-12% Bis-Tris Gels, 1.0 mm, from Invitrogen, (Carlsbad, CA, USA) were used with a NuPAGE MOPS SDS Running Buffer for Bis-Tris Gels.

    Techniques: SDS Page, Purification, Staining

    4-12% Bis-Tris SDS-PAGE gel of cell free extracts of E. coli BL21 (DE3) Gold expressing Sm NOX.

    Journal: Computational and Structural Biotechnology Journal

    Article Title: Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations

    doi: 10.5936/csbj.201402005

    Figure Lengend Snippet: 4-12% Bis-Tris SDS-PAGE gel of cell free extracts of E. coli BL21 (DE3) Gold expressing Sm NOX.

    Article Snippet: For SDS-PAGE NuPAGE® 4-12% Bis-Tris Gels, 1.0 mm, from Invitrogen, (Carlsbad, CA, USA) were used with a NuPAGE MOPS SDS Running Buffer for Bis-Tris Gels.

    Techniques: SDS Page, Expressing

    Summary of pTF.CREGr expression and purification screen. NuPage 4-12% Bis-Tris gels loaded with (A) PageRuler Plus Prestained Protein Ladder and final elutions of various purified pTF.CREGr truncations from small scale protein expression experiments, and (B) PageRuler Plus Prestained Protein Ladder and a final elution of purified Δ31_pTF.CREGr-His6 from a large scale protein expression experiment. CTHF: C-terminal His6 and FLAG tag. NTH: N-terminal His6 tag. L: protein ladder.

    Journal: bioRxiv

    Article Title: Phytotransferrin endocytosis mediates a direct cell surface-to-chloroplast iron trafficking axis in marine diatoms

    doi: 10.1101/806539

    Figure Lengend Snippet: Summary of pTF.CREGr expression and purification screen. NuPage 4-12% Bis-Tris gels loaded with (A) PageRuler Plus Prestained Protein Ladder and final elutions of various purified pTF.CREGr truncations from small scale protein expression experiments, and (B) PageRuler Plus Prestained Protein Ladder and a final elution of purified Δ31_pTF.CREGr-His6 from a large scale protein expression experiment. CTHF: C-terminal His6 and FLAG tag. NTH: N-terminal His6 tag. L: protein ladder.

    Article Snippet: 1 μg soluble protein and 0.5 μL resuspended insoluble protein fraction were resolved on NuPage 4-12% Bis-Tris 1.5 mm gels (Thermo Fischer Scientific, Catalog #NP0335BOX), wet transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fischer Scientific, Catalog #LC2005) and visualized with WesternBreeze™ Chemiluminescent Kit, anti-rabbit (Thermo Fischer Scientific, Catalog #WB7106).

    Techniques: Expressing, Purification, FLAG-tag

    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

    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

    ApoD and LCAT proteins, but not ApoA1 protein, were present in keratocytes. In a Western blot assay, lysates from keratocytes, dermal fibroblasts and HepG2 cells were solubilized and loaded onto 4 to 12% NuPAGE Bis-Tris gradient gels. After electrophoresis, resolved proteins were transferred onto a nitrocellulose membrane and incubated with antibodies against either ApoA1 ( a ), ApoD ( c ) or LCAT ( e ). Anti-β-Actin served as a loading control ( b , d , f ).

    Journal: Biomolecules

    Article Title: LCAT, ApoD, and ApoA1 Expression and Review of Cholesterol Deposition in the Cornea

    doi: 10.3390/biom9120785

    Figure Lengend Snippet: ApoD and LCAT proteins, but not ApoA1 protein, were present in keratocytes. In a Western blot assay, lysates from keratocytes, dermal fibroblasts and HepG2 cells were solubilized and loaded onto 4 to 12% NuPAGE Bis-Tris gradient gels. After electrophoresis, resolved proteins were transferred onto a nitrocellulose membrane and incubated with antibodies against either ApoA1 ( a ), ApoD ( c ) or LCAT ( e ). Anti-β-Actin served as a loading control ( b , d , f ).

    Article Snippet: The gels were then transferred at 4 °C to nitrocellulose membranes using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories #1703930) in NuPAGE Transfer Buffer (25 mM Bicine, 25 mM Bis-tris, 1 mM ethylenediaminetetraacetic acid, 0.05 mM chlorobutanol, 20% methanol, pH 7.2) (Invitrogen # NP0006) with a PowerPack Basic Power Supply.

    Techniques: Western Blot, Electrophoresis, Incubation

    Silver-stained SDS-PAGE gel (20% gradient NuPAGE system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ].

    Journal: Microorganisms

    Article Title: Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology

    doi: 10.3390/microorganisms6010003

    Figure Lengend Snippet: Silver-stained SDS-PAGE gel (20% gradient NuPAGE system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ].

    Article Snippet: Furthermore, the accompanying NuPAGE MES SDS running buffer (Life Technologies) was used.

    Techniques: Staining, SDS Page, Purification, Expressing, Recombinant

    Silver-stained SDS-PAGE gel (20% gradient NuPAGE system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ). A pre-stained ladder was loaded (6 μL), and 23 μL of each sample was loaded into each lane. The predicted molecular weight of RodA is 15.2 kDa. The identity of these bands as RodA at these locations was confirmed by mass spectrometry in [ 38 ]. These are likely glycoforms of the protein [ 57 ].

    Journal: Microorganisms

    Article Title: Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology

    doi: 10.3390/microorganisms6010003

    Figure Lengend Snippet: Silver-stained SDS-PAGE gel (20% gradient NuPAGE system) of the purification fractions from the culture supernatant of P. pastoris KM71H transformants expressing recombinant hydrophobin RodA ( A. fumigatus ). A pre-stained ladder was loaded (6 μL), and 23 μL of each sample was loaded into each lane. The predicted molecular weight of RodA is 15.2 kDa. The identity of these bands as RodA at these locations was confirmed by mass spectrometry in [ 38 ]. These are likely glycoforms of the protein [ 57 ].

    Article Snippet: Nu-PAGE Bis-Tris Mini Gels (4–12% gradient) and NuPAGE LDS sample buffer with NuPAGE reducing agent (Life Technologies, Carlsbad, CA, USA) were used for the analysis of samples derived from the 500 mL cultures and bioreactor productions.

    Techniques: Staining, SDS Page, Purification, Expressing, Recombinant, Molecular Weight, Mass Spectrometry

    ( A ) Coomassie-stained (G-250) SDS-PAGE gel (20% gradient NuPAGE system) of the recombinant hydrophobins purified from bioreactor cultivation and ( B ) Western blot of SDS-PAGE gel against hydrophobin 6His-tag. Transferred gel was run under the same conditions as those for ( A ). A pre-stained ladder was loaded (3 μL) along with 23 μL of each sample. Red arrows in ( A ) indicate bands that are detected by the Western blot in ( B ). The green arrows indicate locations where CMil3 is believed to reside on the gel despite testing negatively in the Western Blot of ( B ). The green arrow at the top of the well indicates that there may be aggregation of the protein prior to or during the running of the sample on the gel. The predicted molecular weights of the proteins are as follows: RodA 15.2 kDa, CMil1 12.8 kDa, CMil2 10.0 kDa, and CMil3 9.7 kDa. Glycosylation likely accounts for the higher-than-expected position of RodA on the gel (between 15 and 20 kDa) [ 38 , 57 ].

    Journal: Microorganisms

    Article Title: Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology

    doi: 10.3390/microorganisms6010003

    Figure Lengend Snippet: ( A ) Coomassie-stained (G-250) SDS-PAGE gel (20% gradient NuPAGE system) of the recombinant hydrophobins purified from bioreactor cultivation and ( B ) Western blot of SDS-PAGE gel against hydrophobin 6His-tag. Transferred gel was run under the same conditions as those for ( A ). A pre-stained ladder was loaded (3 μL) along with 23 μL of each sample. Red arrows in ( A ) indicate bands that are detected by the Western blot in ( B ). The green arrows indicate locations where CMil3 is believed to reside on the gel despite testing negatively in the Western Blot of ( B ). The green arrow at the top of the well indicates that there may be aggregation of the protein prior to or during the running of the sample on the gel. The predicted molecular weights of the proteins are as follows: RodA 15.2 kDa, CMil1 12.8 kDa, CMil2 10.0 kDa, and CMil3 9.7 kDa. Glycosylation likely accounts for the higher-than-expected position of RodA on the gel (between 15 and 20 kDa) [ 38 , 57 ].

    Article Snippet: Nu-PAGE Bis-Tris Mini Gels (4–12% gradient) and NuPAGE LDS sample buffer with NuPAGE reducing agent (Life Technologies, Carlsbad, CA, USA) were used for the analysis of samples derived from the 500 mL cultures and bioreactor productions.

    Techniques: Staining, SDS Page, Recombinant, Purification, Western Blot

    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p

    Journal: Oncotarget

    Article Title: Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas

    doi: 10.18632/oncotarget.7434

    Figure Lengend Snippet: CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p

    Article Snippet: Western analysis Whole cell extracts (100 μg), either transfected with indicated miRNAs or mock transfected, were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer (Invitrogen, Grand Island, NY).

    Techniques: Expressing, Transfection, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page

    A) SDS reduced 10–20% Tricine gel of mojastin disintegrins. The gel was run under reducing condition with 1X Tricine SDS running buffer using an XCell SureLock Mini cell at 125V for 90 min. The gel was stained with Simply Blue Safe Stain for 1 h and destained overnight with Milli-Q water. Lane 1: SeeBlue Plus2 markers; Lane 2: native mojastin; Lane 3: r-mojastin 1; and Lane 4: r-mojastin 1-GST. B) MALDI-TOF mass spectrums of mojastin disintegrins. The samples were run in a linear mode using an ion source 1 of 20.00 kV, ion source 2 of 18.40 kV, a lens of 9.00 kV, and a pulse ion extraction of 350 ns on a Bruker Daltonics MALDI-TOF-TOF. B) r-mojastin 1; C) native mojastin; and D) r-mojastin 1-GST.

    Journal: Thrombosis research

    Article Title: CLONING, EXPRESSION, AND HEMOSTATIC ACTIVITIES OF A DISINTEGRIN, r-MOJASTIN 1, FROM THE MOHAVE RATTLESNAKE (Crotalus scutulatus scutulatus)

    doi: 10.1016/j.thromres.2010.06.006

    Figure Lengend Snippet: A) SDS reduced 10–20% Tricine gel of mojastin disintegrins. The gel was run under reducing condition with 1X Tricine SDS running buffer using an XCell SureLock Mini cell at 125V for 90 min. The gel was stained with Simply Blue Safe Stain for 1 h and destained overnight with Milli-Q water. Lane 1: SeeBlue Plus2 markers; Lane 2: native mojastin; Lane 3: r-mojastin 1; and Lane 4: r-mojastin 1-GST. B) MALDI-TOF mass spectrums of mojastin disintegrins. The samples were run in a linear mode using an ion source 1 of 20.00 kV, ion source 2 of 18.40 kV, a lens of 9.00 kV, and a pulse ion extraction of 350 ns on a Bruker Daltonics MALDI-TOF-TOF. B) r-mojastin 1; C) native mojastin; and D) r-mojastin 1-GST.

    Article Snippet: The disintegrins were subjected to electrophoresis by using a pre-cast 10–20% Tricine gel [ ] in an Xcell SureLock Mini-Cell (Invitrogen Life Technologies, USA).

    Techniques: Staining