hrp-conjugated secondary antibody Search Results


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  • 86
    Santa Cruz Biotechnology secondary antibody hrp conjugated secondary antibodies
    Treatment with SXR activators increases expression of p53 and p53 target genes . Total RNA was isolated from A) MCF-7 cells or B) ZR75-1 cells grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO) for 72 hours and 24 hours respectively. RNA was reverse transcribed and analyzed by QRT-PCR using primers for human p53, p21, BAX and PUMA. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. These results were replicated in at least three independent experiments. *represents P ≤ .05, **represents P ≤ .01 and # represents P ≤ .001 (by one-way ANOVA analysis). C) SXR activation causes p53 accumulation. Cell lysates made from C) MCF-7 and D) ZR-75-1 cells treated with SXR agonists (10 μM) or solvent controls for 72 and 24 hours respectively were subjected to Western blot analysis using p53 antibody (FL-393 <t>HRP,</t> Santa <t>Cruz</t> Inc.). Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin (CAMP) 24 hour treatment was used as a positive control for p53 induction. The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech). The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR-75-1 is because of gel tearing at the time of transferring.
    Secondary Antibody Hrp Conjugated Secondary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam hrp conjugated secondary antibodies
    Treatment with SXR activators increases expression of p53 and p53 target genes . Total RNA was isolated from A) MCF-7 cells or B) ZR75-1 cells grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO) for 72 hours and 24 hours respectively. RNA was reverse transcribed and analyzed by QRT-PCR using primers for human p53, p21, BAX and PUMA. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. These results were replicated in at least three independent experiments. *represents P ≤ .05, **represents P ≤ .01 and # represents P ≤ .001 (by one-way ANOVA analysis). C) SXR activation causes p53 accumulation. Cell lysates made from C) MCF-7 and D) ZR-75-1 cells treated with SXR agonists (10 μM) or solvent controls for 72 and 24 hours respectively were subjected to Western blot analysis using p53 antibody (FL-393 <t>HRP,</t> Santa <t>Cruz</t> Inc.). Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin (CAMP) 24 hour treatment was used as a positive control for p53 induction. The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech). The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR-75-1 is because of gel tearing at the time of transferring.
    Hrp Conjugated Secondary Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1075 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare hrp conjugated secondary antibody
    Native EAG and <t>CMG</t> coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by <t>HRP-conjugated</t> anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.
    Hrp Conjugated Secondary Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 4831 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Jackson Immuno hrp conjugated secondary antibodies
    dCIP4 and Wsp inhibit Gbb secretion. A1–D2 , Confocal images of NMJs 6/7 stained for extracellular <t>Gbb-GFP</t> (red) and <t>HRP</t> (blue) in BG57-GAL4 /+ ( A1 , A2 ), BG57-GAL4 / UAS-gbb-GFP ( B1 , B2 ), BG57-GAL4,Df(3L)ED4342 / UAS-gbb-GFP,dcip4 1 ( C1 , C2 ), and BG57-GAL4
    Hrp Conjugated Secondary Antibodies, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 4194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SouthernBiotech hrp conjugated secondary antibody
    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% <t>glutaraldehyde/PBS.</t> An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an <t>HRP-conjugated</t> secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Hrp Conjugated Secondary Antibody, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 92/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bioworld Antibodies hrp conjugated secondary antibody
    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% <t>glutaraldehyde/PBS.</t> An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an <t>HRP-conjugated</t> secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Hrp Conjugated Secondary Antibody, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega horseradish peroxidise conjugated secondary antibodies
    Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish <t>peroxidise-conjugated</t> secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P
    Horseradish Peroxidise Conjugated Secondary Antibodies, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies secondary hrp conjugated antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Secondary Hrp Conjugated Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beyotime hrp conjugated secondary antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Hrp Conjugated Secondary Antibodies, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abmart hrp conjugated secondary antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Hrp Conjugated Secondary Antibodies, supplied by Abmart, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    WuXi AppTec hrp conjugated secondary antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Hrp Conjugated Secondary Antibodies, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech hrp conjugated secondary antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Hrp Conjugated Secondary Antibodies, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    The Jackson Laboratory hrp conjugated secondary antibodies
    Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with <t>streptavidin-HRP</t> to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value
    Hrp Conjugated Secondary Antibodies, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc hrp conjugated secondary antibody
    H 2 O 2 increases the cytoplasmic La level. Western blots showing the levels of the La protein and the internal loading control β -tubulin/lamin B1 in the cytoplasmic S10, nuclear extracts and total lysates, in Huh7 cells treated with doses of H 2 O 2 , as indicated, for 1 h. The following concentrations were used: anti-La antibody <t>SW5</t> (1 : 5000); β -tubulin (1 : 5000; clone TUB2.1, Sigma); lamin B1 (1 : 1000; D9V6H, Cell Signaling); <t>HRP-conjugated</t> anti-mouse antibody (1 : 5000; Cell Signaling); HRP-conjugated anti-rabbit antibody (1 : 500; Cell Signaling). The levels of La were quantified using ImageJ, normalized against β -tubulin (for S10 fractions and total lysates)/lamin B1 (for nuclear fractions) and expressed as fold increase relative to the 0 µM H 2 O 2 control.
    Hrp Conjugated Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 2068 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer hrp conjugated secondary antibody
    H 2 O 2 increases the cytoplasmic La level. Western blots showing the levels of the La protein and the internal loading control β -tubulin/lamin B1 in the cytoplasmic S10, nuclear extracts and total lysates, in Huh7 cells treated with doses of H 2 O 2 , as indicated, for 1 h. The following concentrations were used: anti-La antibody <t>SW5</t> (1 : 5000); β -tubulin (1 : 5000; clone TUB2.1, Sigma); lamin B1 (1 : 1000; D9V6H, Cell Signaling); <t>HRP-conjugated</t> anti-mouse antibody (1 : 5000; Cell Signaling); HRP-conjugated anti-rabbit antibody (1 : 500; Cell Signaling). The levels of La were quantified using ImageJ, normalized against β -tubulin (for S10 fractions and total lysates)/lamin B1 (for nuclear fractions) and expressed as fold increase relative to the 0 µM H 2 O 2 control.
    Hrp Conjugated Secondary Antibody, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 536 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Zhongshan Golden Bridge Company hrp conjugated secondary antibody
    Xenopus golph2 is expressed in epithelial cells. Stage 46/47 embryos were fixed, embedded in paraffin, sectioned to a thickness of 3 µm. Endogenous golph2 was labeled with the primary antibody <t>10F12,</t> followed by anti-mouse <t>HRP-conjugated</t> secondary antibody and stained with DAB solution; hematoxylin nuclear counterstaining. nt, neural tube; so, somites; pn, pronephros; oe, esophagus; lu, lung; lv, liver; pa, pancreas; st, stomach; i, intestine; s, skin.
    Hrp Conjugated Secondary Antibody, supplied by Zhongshan Golden Bridge Company, used in various techniques. Bioz Stars score: 92/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Treatment with SXR activators increases expression of p53 and p53 target genes . Total RNA was isolated from A) MCF-7 cells or B) ZR75-1 cells grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO) for 72 hours and 24 hours respectively. RNA was reverse transcribed and analyzed by QRT-PCR using primers for human p53, p21, BAX and PUMA. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. These results were replicated in at least three independent experiments. *represents P ≤ .05, **represents P ≤ .01 and # represents P ≤ .001 (by one-way ANOVA analysis). C) SXR activation causes p53 accumulation. Cell lysates made from C) MCF-7 and D) ZR-75-1 cells treated with SXR agonists (10 μM) or solvent controls for 72 and 24 hours respectively were subjected to Western blot analysis using p53 antibody (FL-393 HRP, Santa Cruz Inc.). Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin (CAMP) 24 hour treatment was used as a positive control for p53 induction. The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech). The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR-75-1 is because of gel tearing at the time of transferring.

    Journal: BMC Cancer

    Article Title: Activation of the steroid and xenobiotic receptor, SXR, induces apoptosis in breast cancer cells

    doi: 10.1186/1471-2407-9-3

    Figure Lengend Snippet: Treatment with SXR activators increases expression of p53 and p53 target genes . Total RNA was isolated from A) MCF-7 cells or B) ZR75-1 cells grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO) for 72 hours and 24 hours respectively. RNA was reverse transcribed and analyzed by QRT-PCR using primers for human p53, p21, BAX and PUMA. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. These results were replicated in at least three independent experiments. *represents P ≤ .05, **represents P ≤ .01 and # represents P ≤ .001 (by one-way ANOVA analysis). C) SXR activation causes p53 accumulation. Cell lysates made from C) MCF-7 and D) ZR-75-1 cells treated with SXR agonists (10 μM) or solvent controls for 72 and 24 hours respectively were subjected to Western blot analysis using p53 antibody (FL-393 HRP, Santa Cruz Inc.). Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin (CAMP) 24 hour treatment was used as a positive control for p53 induction. The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech). The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR-75-1 is because of gel tearing at the time of transferring.

    Article Snippet: The primary antibody incubation was followed by 1 hr incubation at room temperature with HRP-conjugated secondary antibody (1:10,000; Santa Cruz Biotechnology Inc., USA).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Activation Assay, Western Blot, Stripping Membranes, Positive Control, Software, Transferring

    Native EAG and CMG coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by HRP-conjugated anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.

    Journal: The Journal of Neuroscience

    Article Title: Camguk/CASK Enhances Ether-Á-Go-Go Potassium Current by a Phosphorylation-Dependent Mechanism

    doi: 10.1523/JNEUROSCI.4566-04.2005

    Figure Lengend Snippet: Native EAG and CMG coimmunoprecipitate from Drosophila extracts. Protein extracts were prepared from the nervous system, imaginal discs, and body muscle fibers of third-instar larvae. CMG antisera were used to immunopreciptate CMG and associating proteins from extracts prepared from the indicated genotypes. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with either CMG ( A ) or EAG (NT) antisera ( B , right), followed by HRP-conjugated anti-guinea pig or anti-rabbit IgG, respectively. For comparison, input extract probed with EAG (NT) antisera is shown in the left panel of B . Respective loads were 5, 10, and 20 μl for extracts, immunoprecipitated proteins, and coimmunoprecipitated proteins, respectively (see Materials and Methods). Similar results were obtained in five experiments. Bands were visualized using ECL. WT, Wild type; IP, immunoprecipitation.

    Article Snippet: Blots were blocked (5% dry milk in TBS) and washed, probed with either CMG or EAG (CT) sera (1:1000 and 1:2000, respectively) followed by HRP-conjugated secondary antibody (1:2000), and visualized by ECL (Amersham Biosciences).

    Techniques: SDS Page, Immunoprecipitation

    dCIP4 and Wsp inhibit Gbb secretion. A1–D2 , Confocal images of NMJs 6/7 stained for extracellular Gbb-GFP (red) and HRP (blue) in BG57-GAL4 /+ ( A1 , A2 ), BG57-GAL4 / UAS-gbb-GFP ( B1 , B2 ), BG57-GAL4,Df(3L)ED4342 / UAS-gbb-GFP,dcip4 1 ( C1 , C2 ), and BG57-GAL4

    Journal: The Journal of Neuroscience

    Article Title: dCIP4 (Drosophila Cdc42-Interacting Protein 4) Restrains Synaptic Growth by Inhibiting the Secretion of the Retrograde Glass Bottom Boat Signal

    doi: 10.1523/JNEUROSCI.0256-10.2010

    Figure Lengend Snippet: dCIP4 and Wsp inhibit Gbb secretion. A1–D2 , Confocal images of NMJs 6/7 stained for extracellular Gbb-GFP (red) and HRP (blue) in BG57-GAL4 /+ ( A1 , A2 ), BG57-GAL4 / UAS-gbb-GFP ( B1 , B2 ), BG57-GAL4,Df(3L)ED4342 / UAS-gbb-GFP,dcip4 1 ( C1 , C2 ), and BG57-GAL4

    Article Snippet: The following antibodies were used in this study: rat anti-dCIP4 (1:1000), guinea pig anti-Wsp (a kind gift from Sven Bogdan, Universität M ̈ unster, M ̈ unster, Germany; 1:1000), rabbit anti-dynamin (a kind gift from Mani Ramaswami, Trinity College Dublin, Dublin, Ireland; 1:1000), rabbit anti-Actin (Sigma; 1:1000), rabbit anti-Myc (Cell Signaling Technology; 1:1000), mouse anti-GFP (Roche; 1:1000), and HRP-conjugated secondary antibodies (Jackson ImmunoResearch; 1:5000).

    Techniques: Staining

    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Article Snippet: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

    Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Article Snippet: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

    Techniques: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the plate, were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole, and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the plate, were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole, and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Article Snippet: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

    Techniques: Inhibition, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish peroxidise-conjugated secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P

    Journal: Scientific Reports

    Article Title: Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo

    doi: 10.1038/s41598-017-07494-0

    Figure Lengend Snippet: Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish peroxidise-conjugated secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P

    Article Snippet: Rabbit polyclonal anti-bee defensin-1 (Def-1) was purchased from GenCust Europe (Luxembourg), and horseradish peroxidise-conjugated secondary antibodies were obtained from Promega (USA).

    Techniques: Purification, Incubation, Zymography, Activity Assay, High Performance Liquid Chromatography, Flow Cytometry, SDS Page, Western Blot, Blocking Assay

    Binding of recombinant CD2831 full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by HRP-conjugated-secondary antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.

    Journal: Scientific Reports

    Article Title: Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis

    doi: 10.1038/s41598-019-42000-8

    Figure Lengend Snippet: Binding of recombinant CD2831 full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by HRP-conjugated-secondary antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.

    Article Snippet: After blocking with PBS containing 0.05% Tween-20 (PBS-T) and 10% (w/v) milk (Sigma, USA), proteins were detected with rabbit anti-CD2831 serum diluted 1:1000 for 1 hour at RT, followed by the HRP-conjugated secondary antibodies (Dako, Denmark) diluted 1:2000 for 1 hour at RT.

    Techniques: Binding Assay, Recombinant, Immunofluorescence, Produced, Staining, Cell Culture, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay, Purification, Negative Control

    Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with streptavidin-HRP to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value

    Journal: PLoS ONE

    Article Title: Degradation of Internalized ?v?5 Integrin Is Controlled by uPAR Bound uPA: Effect on ?1 Integrin Activity and ?-SMA Stress Fiber Assembly

    doi: 10.1371/journal.pone.0033915

    Figure Lengend Snippet: Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with streptavidin-HRP to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value

    Article Snippet: HRP-conjugated anti-Streptavidin antibody and all HRP-conjugated secondary antibodies were from Jackson Laboratories.

    Techniques: Expressing, Flow Cytometry, Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Real-time Polymerase Chain Reaction

    H 2 O 2 increases the cytoplasmic La level. Western blots showing the levels of the La protein and the internal loading control β -tubulin/lamin B1 in the cytoplasmic S10, nuclear extracts and total lysates, in Huh7 cells treated with doses of H 2 O 2 , as indicated, for 1 h. The following concentrations were used: anti-La antibody SW5 (1 : 5000); β -tubulin (1 : 5000; clone TUB2.1, Sigma); lamin B1 (1 : 1000; D9V6H, Cell Signaling); HRP-conjugated anti-mouse antibody (1 : 5000; Cell Signaling); HRP-conjugated anti-rabbit antibody (1 : 500; Cell Signaling). The levels of La were quantified using ImageJ, normalized against β -tubulin (for S10 fractions and total lysates)/lamin B1 (for nuclear fractions) and expressed as fold increase relative to the 0 µM H 2 O 2 control.

    Journal: The Journal of General Virology

    Article Title: Hydrogen peroxide induces La cytoplasmic shuttling and increases hepatitis C virus internal ribosome entry site-dependent translation

    doi: 10.1099/jgv.0.000556

    Figure Lengend Snippet: H 2 O 2 increases the cytoplasmic La level. Western blots showing the levels of the La protein and the internal loading control β -tubulin/lamin B1 in the cytoplasmic S10, nuclear extracts and total lysates, in Huh7 cells treated with doses of H 2 O 2 , as indicated, for 1 h. The following concentrations were used: anti-La antibody SW5 (1 : 5000); β -tubulin (1 : 5000; clone TUB2.1, Sigma); lamin B1 (1 : 1000; D9V6H, Cell Signaling); HRP-conjugated anti-mouse antibody (1 : 5000; Cell Signaling); HRP-conjugated anti-rabbit antibody (1 : 500; Cell Signaling). The levels of La were quantified using ImageJ, normalized against β -tubulin (for S10 fractions and total lysates)/lamin B1 (for nuclear fractions) and expressed as fold increase relative to the 0 µM H 2 O 2 control.

    Article Snippet: Cells grown on chamber slides (Falcon) were fixed in ice-cold methanol for 1 h. After blocking for 1 h in 1 % horse serum/2 % BSA/PBS, cells were incubated with anti-La antibody SW5 (1 : 10) or isotypic control, and then HRP-conjugated secondary antibody (1 : 50; Cell Signaling) for 1 h each in blocking buffer.

    Techniques: Western Blot

    Xenopus golph2 is expressed in epithelial cells. Stage 46/47 embryos were fixed, embedded in paraffin, sectioned to a thickness of 3 µm. Endogenous golph2 was labeled with the primary antibody 10F12, followed by anti-mouse HRP-conjugated secondary antibody and stained with DAB solution; hematoxylin nuclear counterstaining. nt, neural tube; so, somites; pn, pronephros; oe, esophagus; lu, lung; lv, liver; pa, pancreas; st, stomach; i, intestine; s, skin.

    Journal: PLoS ONE

    Article Title: Xenopus as a Model System for the Study of GOLPH2/GP73 Function: Xenopus golph2 Is Required for Pronephros Development

    doi: 10.1371/journal.pone.0038939

    Figure Lengend Snippet: Xenopus golph2 is expressed in epithelial cells. Stage 46/47 embryos were fixed, embedded in paraffin, sectioned to a thickness of 3 µm. Endogenous golph2 was labeled with the primary antibody 10F12, followed by anti-mouse HRP-conjugated secondary antibody and stained with DAB solution; hematoxylin nuclear counterstaining. nt, neural tube; so, somites; pn, pronephros; oe, esophagus; lu, lung; lv, liver; pa, pancreas; st, stomach; i, intestine; s, skin.

    Article Snippet: Endogenous golph2 was localized using the antibody 10F12 followed by incubation with a Cy3-conjugated secondary antibody (Chemicon, USA) or HRP-conjugated secondary antibody (Zhongshan Golden Bridge, China).

    Techniques: Labeling, Staining