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  • 99
    Thermo Fisher nupage
    IP 7 pyrophosphorylates RNA Pol I subunits ( A ) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β- 32 P]IP 7 . Proteins were resolved using <t>NuPAGE</t> and transferred to a <t>PVDF</t> membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). ( B ) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in ( A ). ( C ) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). ( D–F ) Pyrophosphorylation, as in ( A ), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. ( G ) Pyrophosphorylation, as in ( A ), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in ( A ), ( F ) and ( G ) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels ( A ) and ( F ) indicate the removal of non-essential lanes from a single original gel.
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    99
    Thermo Fisher nupage lds
    BoTest cell-free light chain activity assay. (A) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.5 pM—1.25 nM) were incubated with 200 nM BoTest Reporter (VAMP-2 [33–94] flanked by N-terminal cyan fluorescent protein [CFP] and C-terminal yellow fluorescent protein [YFP]) for 18 hours at 30°C. Fluorescence intensity at 526 nm (YFP) and 470 nm (CFP) was determined using a BioTek Synergy HT plate reader. A diminishing 526/470 nm relative fluorescent unit (RFU) ratio indicates substrate cleavage. Data were fitted to a four-parameter equation. (B) The concentration of each toxin required for 50% maximum inhibition of 526/470 RFU ratio (pIC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 6 (BoNT/B1) or n = 3 (rBoNT/B1 and rBoNT/B1 (S201P) ) independent experiments, each performed in duplicate. (C) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.1 pM—10 nM) were incubated with 2 μM VAMP-1 (2–96)-GFP at 37°C for 24 hr. Reactions were terminated by addition of <t>NuPAGE</t> <t>LDS</t> buffer, 1 mM DTT and separated by SDS-PAGE on 12% Bis-tris gels. Gels were stained and densitometry performed to determine the percentage substrate cleavage of each sample. Data were fitted to a four-parameter equation. (D) The concentration of each toxin required for 50% maximum substrate cleavage (pEC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 3 independent experiments, each performed in triplicate.Significant differences in potency are denoted by **(P
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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).
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    90
    Thermo Fisher nupage gels
    Western blot analysis of purified vaccine and wtRV virions and viral proteins in the infected HUVEC. Vero and HUVEC were infected with <t>RA27/3</t> and RV-Dz at MOI = 5. Virions were pelleted from the culture media by ultracentrifugation and resuspended in an SDS-sample buffer. The cell monolayers were washed with PBS and proteins were then extracted with RIPA buffer. Proteins were separated by a 4–12% <t>NuPage</t> gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb to identify RV structural proteins. The blots were also probed with β-actin MAb to demonstrate equal protein loading for the analysis of the cell lysates and show purity of the pelleted virions. Representative results of two independent experiments are shown.
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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.
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    99
    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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    99
    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 3504 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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    92
    Thermo Fisher nupage mops 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 Mops Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 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
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    99
    Thermo Fisher nupage antioxidant
    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
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    Image Search Results


    IP 7 pyrophosphorylates RNA Pol I subunits ( A ) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). ( B ) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in ( A ). ( C ) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). ( D–F ) Pyrophosphorylation, as in ( A ), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. ( G ) Pyrophosphorylation, as in ( A ), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in ( A ), ( F ) and ( G ) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels ( A ) and ( F ) indicate the removal of non-essential lanes from a single original gel.

    Journal: Biochemical Journal

    Article Title: Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae

    doi: 10.1042/BJ20140798

    Figure Lengend Snippet: IP 7 pyrophosphorylates RNA Pol I subunits ( A ) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). ( B ) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in ( A ). ( C ) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). ( D–F ) Pyrophosphorylation, as in ( A ), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. ( G ) Pyrophosphorylation, as in ( A ), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in ( A ), ( F ) and ( G ) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels ( A ) and ( F ) indicate the removal of non-essential lanes from a single original gel.

    Article Snippet: The pyrophosphorylation reaction was performed with proteins bound to glutathione beads in the presence of IP7 reaction buffer (25 mM Hepes, pH 7.4, 50 mM NaCl, 6 mM MgCl2 , 1 mM DTT) and 1 μCi of 5[β-32 P]IP7 (120 Ci/mmol) at 37°C for 15 min. To the reaction mixture, LDS sample buffer (Invitrogen) was added and incubated at 95°C for 5 min. Proteins were resolved on a 4–12% gradient gel (Invitrogen) by NuPAGE (Life Technologies) and transferred to a PVDF membrane (GE Life Sciences).

    Techniques: Purification, Incubation, Western Blot, Sequencing

    Detection of Jun in walleye cell nuclear extracts. Recombinant human Jun (rhAP1), A431 cell lysate, HeLa cell nuclear extract (NE), and W12 cell nuclear extract were separated on a NuPage 12% Bis-Tris gel and transferred to an Immobilon membrane. The blot was probed with rabbit anti-Jun antiserum that recognizes the DNA-binding domain of avian Jun.

    Journal: Journal of Virology

    Article Title: Identification and Characterization of cis-Acting Elements Residing in the Walleye Dermal Sarcoma Virus Promoter

    doi: 10.1128/JVI.78.14.7590-7601.2004

    Figure Lengend Snippet: Detection of Jun in walleye cell nuclear extracts. Recombinant human Jun (rhAP1), A431 cell lysate, HeLa cell nuclear extract (NE), and W12 cell nuclear extract were separated on a NuPage 12% Bis-Tris gel and transferred to an Immobilon membrane. The blot was probed with rabbit anti-Jun antiserum that recognizes the DNA-binding domain of avian Jun.

    Article Snippet: Fifty nanograms of recombinant human Jun (rhAP1; Promega), 20 μg of A431 cell lysate (Upstate Biotechnology), 10 μg of HeLa cell nuclear extract, and 10 μg of W12 cell nuclear extract were separated on a 12% NuPAGE gel (Invitrogen) in MOPS (3-[ N -morpholino]propanesulfonic acid) running buffer (50 mM MOPS, 50 mM Tris base, 3.5 mM sodium dodecyl sulfate, 1 mM EDTA) at 200 V for 70 min.

    Techniques: Recombinant, Binding Assay

    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

    Determination of μ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ); μ-OR was recognized by C-terminus-oriented Ab C-20 (sc-7488-R). When resolved under non-dissociated conditions ( A and B ), Ab C-20 distinguished the single protein with M w ≈ 60 kDa. The average intensity of this immunoblot signal was not significantly different when compared in control (— M10) and morphine-treated (+M10) PNS samples (NS, p > 0.05). Resolution under dissociated conditions ( C and D ) revealed the presence of multiple protein bands exhibiting a wide range of M w ≈ 20–90 kDa. These immunoblot signals were also unchanged by morphine (NS, p > 0.05). The results (upper columns) represent the average signal of five immunoblots, each performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the control and morphine-treated PNS samples was analyzed by Student´s t -test using GraphPad Prizm4 . In the lower panels, typical immunoblots are presented.

    Journal: PLoS ONE

    Article Title: Determination of μ-, δ- and κ-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal

    doi: 10.1371/journal.pone.0186797

    Figure Lengend Snippet: Determination of μ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ); μ-OR was recognized by C-terminus-oriented Ab C-20 (sc-7488-R). When resolved under non-dissociated conditions ( A and B ), Ab C-20 distinguished the single protein with M w ≈ 60 kDa. The average intensity of this immunoblot signal was not significantly different when compared in control (— M10) and morphine-treated (+M10) PNS samples (NS, p > 0.05). Resolution under dissociated conditions ( C and D ) revealed the presence of multiple protein bands exhibiting a wide range of M w ≈ 20–90 kDa. These immunoblot signals were also unchanged by morphine (NS, p > 0.05). The results (upper columns) represent the average signal of five immunoblots, each performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the control and morphine-treated PNS samples was analyzed by Student´s t -test using GraphPad Prizm4 . In the lower panels, typical immunoblots are presented.

    Article Snippet: The same samples of PNS were also analyzed by the NuPAGE system (Invitrogen).

    Techniques: SDS Page, Western Blot

    Determination of κ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ); κ-OR was recognized by Ab H-70 (sc-9112). The results (upper columns) represent the average signal of five immunoblot experiments performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the control and morphine-treated PNS samples was analyzed by Student´s t -test using GraphPad Prizm4 . In the lower panels, typical immunoblots are presented (NS, p > 0.05).

    Journal: PLoS ONE

    Article Title: Determination of μ-, δ- and κ-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal

    doi: 10.1371/journal.pone.0186797

    Figure Lengend Snippet: Determination of κ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ); κ-OR was recognized by Ab H-70 (sc-9112). The results (upper columns) represent the average signal of five immunoblot experiments performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the control and morphine-treated PNS samples was analyzed by Student´s t -test using GraphPad Prizm4 . In the lower panels, typical immunoblots are presented (NS, p > 0.05).

    Article Snippet: The same samples of PNS were also analyzed by the NuPAGE system (Invitrogen).

    Techniques: SDS Page, Western Blot

    Determination of δ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ) and δ-OR was recognized by N-terminus-oriented Ab H-60 (sc-9111). Significant down-regulation (↓) of δ-OR isoform with M w ≈ 60 kDa was detected (*, p

    Journal: PLoS ONE

    Article Title: Determination of μ-, δ- and κ-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal

    doi: 10.1371/journal.pone.0186797

    Figure Lengend Snippet: Determination of δ-OR in PNS prepared from FBC of experimental groups (+M10) and (─M10). PNS fractions (20 μg protein per lane) prepared from morphine-treated (+M10) and control (— M10) rats were resolved under non-dissociated (— DTT) or dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide ( A, C) or in 4–12% NuPAGE gels ( B, D ) and δ-OR was recognized by N-terminus-oriented Ab H-60 (sc-9111). Significant down-regulation (↓) of δ-OR isoform with M w ≈ 60 kDa was detected (*, p

    Article Snippet: The same samples of PNS were also analyzed by the NuPAGE system (Invitrogen).

    Techniques: SDS Page

    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

    BoTest cell-free light chain activity assay. (A) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.5 pM—1.25 nM) were incubated with 200 nM BoTest Reporter (VAMP-2 [33–94] flanked by N-terminal cyan fluorescent protein [CFP] and C-terminal yellow fluorescent protein [YFP]) for 18 hours at 30°C. Fluorescence intensity at 526 nm (YFP) and 470 nm (CFP) was determined using a BioTek Synergy HT plate reader. A diminishing 526/470 nm relative fluorescent unit (RFU) ratio indicates substrate cleavage. Data were fitted to a four-parameter equation. (B) The concentration of each toxin required for 50% maximum inhibition of 526/470 RFU ratio (pIC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 6 (BoNT/B1) or n = 3 (rBoNT/B1 and rBoNT/B1 (S201P) ) independent experiments, each performed in duplicate. (C) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.1 pM—10 nM) were incubated with 2 μM VAMP-1 (2–96)-GFP at 37°C for 24 hr. Reactions were terminated by addition of NuPAGE LDS buffer, 1 mM DTT and separated by SDS-PAGE on 12% Bis-tris gels. Gels were stained and densitometry performed to determine the percentage substrate cleavage of each sample. Data were fitted to a four-parameter equation. (D) The concentration of each toxin required for 50% maximum substrate cleavage (pEC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 3 independent experiments, each performed in triplicate.Significant differences in potency are denoted by **(P

    Journal: PLoS ONE

    Article Title: Augmentation of VAMP-catalytic activity of botulinum neurotoxin serotype B does not result in increased potency in physiological systems

    doi: 10.1371/journal.pone.0185628

    Figure Lengend Snippet: BoTest cell-free light chain activity assay. (A) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.5 pM—1.25 nM) were incubated with 200 nM BoTest Reporter (VAMP-2 [33–94] flanked by N-terminal cyan fluorescent protein [CFP] and C-terminal yellow fluorescent protein [YFP]) for 18 hours at 30°C. Fluorescence intensity at 526 nm (YFP) and 470 nm (CFP) was determined using a BioTek Synergy HT plate reader. A diminishing 526/470 nm relative fluorescent unit (RFU) ratio indicates substrate cleavage. Data were fitted to a four-parameter equation. (B) The concentration of each toxin required for 50% maximum inhibition of 526/470 RFU ratio (pIC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 6 (BoNT/B1) or n = 3 (rBoNT/B1 and rBoNT/B1 (S201P) ) independent experiments, each performed in duplicate. (C) BoNT/B1, rBoNT/B1 or rBoNT/B1 (S201P) (0.1 pM—10 nM) were incubated with 2 μM VAMP-1 (2–96)-GFP at 37°C for 24 hr. Reactions were terminated by addition of NuPAGE LDS buffer, 1 mM DTT and separated by SDS-PAGE on 12% Bis-tris gels. Gels were stained and densitometry performed to determine the percentage substrate cleavage of each sample. Data were fitted to a four-parameter equation. (D) The concentration of each toxin required for 50% maximum substrate cleavage (pEC 50 ) was determined from the fitted curves. Data represent the mean ± s.e.m. of n = 3 independent experiments, each performed in triplicate.Significant differences in potency are denoted by **(P

    Article Snippet: The plates were sealed, and incubated at 37°C for 24 h. After incubation, 20 μL 2x NuPAGE LDS sample buffer (Life Technologies, UK) and DTT (final concentration 10 mM) was added to each well and the plate was heated at 70°C for 5 minutes.

    Techniques: Activity Assay, Incubation, Fluorescence, Concentration Assay, Inhibition, 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

    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

    Western blot analysis of purified vaccine and wtRV virions and viral proteins in the infected HUVEC. Vero and HUVEC were infected with RA27/3 and RV-Dz at MOI = 5. Virions were pelleted from the culture media by ultracentrifugation and resuspended in an SDS-sample buffer. The cell monolayers were washed with PBS and proteins were then extracted with RIPA buffer. Proteins were separated by a 4–12% NuPage gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb to identify RV structural proteins. The blots were also probed with β-actin MAb to demonstrate equal protein loading for the analysis of the cell lysates and show purity of the pelleted virions. Representative results of two independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells

    doi: 10.1371/journal.pone.0133267

    Figure Lengend Snippet: Western blot analysis of purified vaccine and wtRV virions and viral proteins in the infected HUVEC. Vero and HUVEC were infected with RA27/3 and RV-Dz at MOI = 5. Virions were pelleted from the culture media by ultracentrifugation and resuspended in an SDS-sample buffer. The cell monolayers were washed with PBS and proteins were then extracted with RIPA buffer. Proteins were separated by a 4–12% NuPage gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb to identify RV structural proteins. The blots were also probed with β-actin MAb to demonstrate equal protein loading for the analysis of the cell lysates and show purity of the pelleted virions. Representative results of two independent experiments are shown.

    Article Snippet: Western blot analysis To analyze protein composition of the pelleted virions, the pellets were suspended in 50 μl of 1X non-reducing SDS sample buffer (BioRad, Richmond, CA), and then 10 μl of the RA27/3 preparation and 1 μl RV-Dz preparation were separated by 4–12% NuPage gels (Invitrogen, Carlsbad, CA).

    Techniques: Western Blot, Purification, Infection

    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

    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

    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

    Enrichment of ABCE1 and eIF3j on 40S complexes and assessment of their role in splitting of 80S ribosomes (A) Total cellular extracts from yeast cells expressing ABCE1-TAP were separated on a sucrose gradient (10–50%) by ultracentrifugation. Proteins of each fraction were analyzed by Western blot using a PAP antibody for the detection of the ABCE1-TAP fusion protein and anti-Nog1 antibody to mark the 60S fraction. (B) Silver stained NuPAGE gel showing elution from affinity purification using ABCE1-TAP performed in Tris or Hepes buffer (see methods for details). (C) Volcano plot showing the fold enrichment of proteins in the elution fraction from the ABCE1-TAP purification in Tris buffer followed by mass spectrometry analysis (LC-MS/MS). The enrichment was calculated relative to an “input” corresponding to an aliquot of the ABCE1-TAP cell lysate used for the affinity purification. It is represented, on the x-axis, as log 2 (LFQ ABCE1-TAP/LFQ input) where LFQ stands for label-free quantification. The y-axis represents the P-value distribution (-log 10 -p -value) calculated using the Student’s t-test for all identified proteins represented by a circle. Proteins above the curved lines show a statistically significant enrichment according to the t-test value. The assay was performed in triplicates. (D) UV-profiles from in vitro splitting reaction triplicates with and without splitting factors (SF; ABCE1, Dom34, Hbs1 and eIF6) and eIF3j. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation. (E) SDS-PAGE of the 40S, 60S and 80S peaks obtained from the i n vitro splitting experiment (D) containing SFs and high amounts of eIF3j. (F) UV-profiles from in vitro “facilitated” splitting reactions. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation. (G) SDS-PAGE of the input factors (eIF3j and ABCE1) as well as 40S and 80S peaks from the “facilitated” splitting experiment.

    Journal: bioRxiv

    Article Title: Structural inventory of native ribosomal ABCE1-43S pre-initiation complexes

    doi: 10.1101/2020.07.09.194902

    Figure Lengend Snippet: Enrichment of ABCE1 and eIF3j on 40S complexes and assessment of their role in splitting of 80S ribosomes (A) Total cellular extracts from yeast cells expressing ABCE1-TAP were separated on a sucrose gradient (10–50%) by ultracentrifugation. Proteins of each fraction were analyzed by Western blot using a PAP antibody for the detection of the ABCE1-TAP fusion protein and anti-Nog1 antibody to mark the 60S fraction. (B) Silver stained NuPAGE gel showing elution from affinity purification using ABCE1-TAP performed in Tris or Hepes buffer (see methods for details). (C) Volcano plot showing the fold enrichment of proteins in the elution fraction from the ABCE1-TAP purification in Tris buffer followed by mass spectrometry analysis (LC-MS/MS). The enrichment was calculated relative to an “input” corresponding to an aliquot of the ABCE1-TAP cell lysate used for the affinity purification. It is represented, on the x-axis, as log 2 (LFQ ABCE1-TAP/LFQ input) where LFQ stands for label-free quantification. The y-axis represents the P-value distribution (-log 10 -p -value) calculated using the Student’s t-test for all identified proteins represented by a circle. Proteins above the curved lines show a statistically significant enrichment according to the t-test value. The assay was performed in triplicates. (D) UV-profiles from in vitro splitting reaction triplicates with and without splitting factors (SF; ABCE1, Dom34, Hbs1 and eIF6) and eIF3j. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation. (E) SDS-PAGE of the 40S, 60S and 80S peaks obtained from the i n vitro splitting experiment (D) containing SFs and high amounts of eIF3j. (F) UV-profiles from in vitro “facilitated” splitting reactions. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation. (G) SDS-PAGE of the input factors (eIF3j and ABCE1) as well as 40S and 80S peaks from the “facilitated” splitting experiment.

    Article Snippet: To control the quality of the affinity purification, a sample of eluates (3%) was separated on acrylamide NuPAGE Novex 4–12% Bis-Tris gels (Life Technologies) and analyzed by silver staining.

    Techniques: Expressing, Western Blot, Staining, Affinity Purification, Purification, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, In Vitro, SDS Page

    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

    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