biotin-conjugated rabbit immunoglobulin Millipore Search Results


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  • 99
    Vector Laboratories biotinylated goat anti rabbit igg
    Biotinylated Goat Anti Rabbit Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 9938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protein a sepharose
    Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of <t>protein-A-Sepharose.</t> The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.
    Protein A Sepharose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg whole molecule biotin antibody
    Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of <t>protein-A-Sepharose.</t> The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.
    Anti Rabbit Igg Whole Molecule Biotin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti biotin antibody
    Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of <t>protein-A-Sepharose.</t> The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.
    Monoclonal Anti Biotin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bovine serum albumin bsa
    Binding of biotinylated, full-length rgC1qR (MF) to immobilized protein A. Biotinylated rgC1qR (∼2 μg/ml) was incubated (60 min, 37°C) on protein A-coated microtiter wells. Binding was detected with AP-STRAV and pNPP substrate. <t>C1q-</t> and <t>BSA-coated</t> microtiter wells served as controls. Values represent means ± SD; n = 3. O.D., optical density.
    Bovine Serum Albumin Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32017 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg whole molecule peroxidase antibody
    Binding of biotinylated, full-length rgC1qR (MF) to immobilized protein A. Biotinylated rgC1qR (∼2 μg/ml) was incubated (60 min, 37°C) on protein A-coated microtiter wells. Binding was detected with AP-STRAV and pNPP substrate. <t>C1q-</t> and <t>BSA-coated</t> microtiter wells served as controls. Values represent means ± SD; n = 3. O.D., optical density.
    Anti Rabbit Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg
    Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit <t>IgG</t> binding (series 1) is shown to demonstrate successful protein A hyperiodination. <t>BSA-coated</t> microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.
    Anti Rabbit Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated secondary antibodies
    Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit <t>IgG</t> binding (series 1) is shown to demonstrate successful protein A hyperiodination. <t>BSA-coated</t> microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.
    Biotinylated Secondary Antibodies, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 10949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti laminin antibody
    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: <t>Laminin;</t> lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p
    Anti Laminin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4043 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated horse anti mouse immunoglobulin g
    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: <t>Laminin;</t> lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p
    Biotinylated Horse Anti Mouse Immunoglobulin G, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories goat anti rabbit
    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: <t>Laminin;</t> lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p
    Goat Anti Rabbit, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno biotin sp affinipure donkey anti rabbit igg
    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: <t>Laminin;</t> lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p
    Biotin Sp Affinipure Donkey Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 1088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated anti mouse igg
    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: <t>Laminin;</t> lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p
    Biotinylated Anti Mouse Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 3254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.

    Article Snippet: Protein-A-Sepharose (Sigma-Aldrich) was then added to the mixtures containing the cell extracts and incubated further for 5 h at 4°C.

    Techniques: Activity Assay, Immunoprecipitation, Incubation, Western Blot, CTL Assay, Negative Control

    Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1 +/+ and PARP-1 -/- cells . ( A ) Immunoprecipitation of the Sp1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. ( B ) Immunoprecipitation of the PARP-1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1 +/+ and PARP-1 -/- cells . ( A ) Immunoprecipitation of the Sp1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. ( B ) Immunoprecipitation of the PARP-1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.

    Article Snippet: Protein-A-Sepharose (Sigma-Aldrich) was then added to the mixtures containing the cell extracts and incubated further for 5 h at 4°C.

    Techniques: Immunoprecipitation, Incubation, SDS-Gel, Western Blot, CTL Assay, Negative Control

    The Δ sdbA mutant does not secrete Sth1 bacteriocins. Secreted bacteriocins were isolated from culture supernatants using Sth 1 -specific rabbit IgG-protein A-Sepharose beads and were detected with a mouse anti-Sth 1 antibody in an enzyme-linked immunosorbent assay (ELISA). (A) Detection of Sth 1 captured from 25 ml of culture supernatant prepared from the parent strain, the Δ sdbA mutant, the sdbA -complemented mutant (SdbA Compl), or uninoculated BHI medium with 5% serum (BHIS) (negative control). (B) Detection of Sth 1 captured from 25 or 100 ml of culture supernatant from the parent strain, the Δ sdbA mutant, or the Δ sdbA mutant induced with exogenous CSP for 30 min. Results are means ± SDs from three experiments. ***, P

    Journal: Journal of Bacteriology

    Article Title: Mutation of the Thiol-Disulfide Oxidoreductase SdbA Activates the CiaRH Two-Component System, Leading to Bacteriocin Expression Shutdown in Streptococcus gordonii

    doi: 10.1128/JB.00800-15

    Figure Lengend Snippet: The Δ sdbA mutant does not secrete Sth1 bacteriocins. Secreted bacteriocins were isolated from culture supernatants using Sth 1 -specific rabbit IgG-protein A-Sepharose beads and were detected with a mouse anti-Sth 1 antibody in an enzyme-linked immunosorbent assay (ELISA). (A) Detection of Sth 1 captured from 25 ml of culture supernatant prepared from the parent strain, the Δ sdbA mutant, the sdbA -complemented mutant (SdbA Compl), or uninoculated BHI medium with 5% serum (BHIS) (negative control). (B) Detection of Sth 1 captured from 25 or 100 ml of culture supernatant from the parent strain, the Δ sdbA mutant, or the Δ sdbA mutant induced with exogenous CSP for 30 min. Results are means ± SDs from three experiments. ***, P

    Article Snippet: The purified antibodies were then irreversibly cross-linked to protein A-Sepharose beads (Sigma-Aldrich) with dimethyl pimelimidate, using standard techniques ( ).

    Techniques: Mutagenesis, Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

    Identification of mouse Sur2 as a specific MAV-1 E1A-interacting protein. (A) Large-scale GST-mE1A pulldown assay. Lane 1, protein molecular size standards; lanes 2 to 4, purified GST, GST-mE1A, and GST-Cter1 fusion proteins, respectively, were bound to glutathione-Sepharose beads without mixing with any mammalian cell nuclear extracts (−); lanes 5 to 7, nuclear extracts from 5 × 10 8  mouse MBMECs were precleared sequentially against glutathione-Sepharose beads and GST beads, and then an equal amount of the precleared mammalian nuclear extracts was added (+) to GST beads, GST-mE1A beads, and GST-Cter1 beads, respectively. Beads were washed, and the bound proteins were eluted off the beads by boiling in protein loading buffer. Proteins binding specifically to GST-mE1A were identified by comparing lane 6 with lanes 3 and 5. The arrowhead shows Sur2 protein. An independent duplicate analysis of both MBMEC and 3T6 cell lines gave similar results, with identification of Sur2 (data not shown). (B) Sur2 protein interacts with GST-mE1A (full-length E1A) in GST pulldowns. GST pulldown assays were performed by incubating the nuclear extracts from 3T6 cells (lanes 1 to 3) or MBMECs (lanes 4 to 6) with GST beads, GST-Cter1 beads, or GST-mE1A beads, as indicated. Monoclonal antibody against Sur2 (BD Pharmingen) (1:1,000) was used for Western blots. Whole-cell lysates (WCL) were used as a positive control (lane 8). Lane 7, protein molecular size standards. The arrowhead shows the Sur2 position. (C) MAV-1 E1A protein interacts with Sur2 in virus-infected cells. MBMECs were mock or MAV-1 infected at an MOI of 5 and harvested at 40 h postinfection. Normal rabbit serum or AKO7-147 (E1A) (both purified by DEAE Affi-Gel blue chromatography) was mixed with whole-cell lysates of MBMECs to carry out the immunoprecipitation. The immunoprecipitates were electrophoresed on 8% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. Monoclonal antibody against Sur2 was used in Western blots. The arrowhead shows the Sur2 position.

    Journal: Journal of Virology

    Article Title: Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

    doi: 10.1128/JVI.78.23.12888-12900.2004

    Figure Lengend Snippet: Identification of mouse Sur2 as a specific MAV-1 E1A-interacting protein. (A) Large-scale GST-mE1A pulldown assay. Lane 1, protein molecular size standards; lanes 2 to 4, purified GST, GST-mE1A, and GST-Cter1 fusion proteins, respectively, were bound to glutathione-Sepharose beads without mixing with any mammalian cell nuclear extracts (−); lanes 5 to 7, nuclear extracts from 5 × 10 8 mouse MBMECs were precleared sequentially against glutathione-Sepharose beads and GST beads, and then an equal amount of the precleared mammalian nuclear extracts was added (+) to GST beads, GST-mE1A beads, and GST-Cter1 beads, respectively. Beads were washed, and the bound proteins were eluted off the beads by boiling in protein loading buffer. Proteins binding specifically to GST-mE1A were identified by comparing lane 6 with lanes 3 and 5. The arrowhead shows Sur2 protein. An independent duplicate analysis of both MBMEC and 3T6 cell lines gave similar results, with identification of Sur2 (data not shown). (B) Sur2 protein interacts with GST-mE1A (full-length E1A) in GST pulldowns. GST pulldown assays were performed by incubating the nuclear extracts from 3T6 cells (lanes 1 to 3) or MBMECs (lanes 4 to 6) with GST beads, GST-Cter1 beads, or GST-mE1A beads, as indicated. Monoclonal antibody against Sur2 (BD Pharmingen) (1:1,000) was used for Western blots. Whole-cell lysates (WCL) were used as a positive control (lane 8). Lane 7, protein molecular size standards. The arrowhead shows the Sur2 position. (C) MAV-1 E1A protein interacts with Sur2 in virus-infected cells. MBMECs were mock or MAV-1 infected at an MOI of 5 and harvested at 40 h postinfection. Normal rabbit serum or AKO7-147 (E1A) (both purified by DEAE Affi-Gel blue chromatography) was mixed with whole-cell lysates of MBMECs to carry out the immunoprecipitation. The immunoprecipitates were electrophoresed on 8% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. Monoclonal antibody against Sur2 was used in Western blots. The arrowhead shows the Sur2 position.

    Article Snippet: The lysates were diluted in E1A binding buffer (125 mM NaCl, 50 mM Tris [pH 7.4], 0.1% NP-40) and then preabsorbed simultaneously against rabbit normal immunoglobulin G (IgG) and protein A-agarose beads (Pharmacia Biotech) by rocking at 4°C for 2 h. The lysates were then incubated with polyclonal antibody against MAV-1 E1A (AKO7-147) ( ) or rabbit normal IgG, followed by incubation with 30 μl of protein A-agarose (Oncogene Research Products).

    Techniques: Purification, Binding Assay, Western Blot, Positive Control, Infection, Chromatography, Immunoprecipitation

    ASFV protein I329L is highly glycosylated and localizes to cell membranes. 2A ) I329L protein distribution is consistent with endoplasmic reticulum localization. Lentivirus-transduced NIH-I329L cells were transfected with plasmid pDsRed2-ER to localize the protein to the ER (red, left) and then co-stained with a rat monoclonal high-affinity antibody against the HA, followed by a donkey affinity-purified anti-rat IgG antibody conjugated with AMCA (left, middle). The colocalization is evident in the merged image (right panel). 2B ) I329L protein distribution is consistent with Golgi localization. The NIH-I329L cells were transfected with plasmid pDsRed-Monomer-Golgi to localize the protein to the Golgi (red, left) and then co-stained with a rat monoclonal high-affinity antibody against the HA, followed by a donkey affinity purified anti-rat IgG antibody conjugated with AMCA (left, middle). The colocalization is evident in the merged image (right panel). 2C ) The I329L protein was immunoprecipitated from lysates from the lentivirus-transduced NIH-I329L cell line with rabbit anti-HA antibodies bound to protein G beads. The bound protein was eluted and digested with EndoH (a) or PNGase (c) and examined by SDS-PAGE. Controls samples were treated with only EndoH buffer (b) or only PNGase buffer (d). 2D ) I329L protein is expressed at the cell surface. Biotinylated surface proteins from biotynylated NIH-I329L cells were collected with streptavidin beads, loaded onto a 10% SDS-PAGE gel and transferred to PVDF membranes, and I329L was revealed with a rat monoclonal antibody against HA conjugated with HRP (band in lane NIHI329L on the left). Lane NIHeGFP represents NIH-3T3 cells transduced with the empty lentivirus. Details are in Materials and methods

    Journal: Archives of Virology

    Article Title: A novel TLR3 inhibitor encoded by African swine fever virus (ASFV)

    doi: 10.1007/s00705-010-0894-7

    Figure Lengend Snippet: ASFV protein I329L is highly glycosylated and localizes to cell membranes. 2A ) I329L protein distribution is consistent with endoplasmic reticulum localization. Lentivirus-transduced NIH-I329L cells were transfected with plasmid pDsRed2-ER to localize the protein to the ER (red, left) and then co-stained with a rat monoclonal high-affinity antibody against the HA, followed by a donkey affinity-purified anti-rat IgG antibody conjugated with AMCA (left, middle). The colocalization is evident in the merged image (right panel). 2B ) I329L protein distribution is consistent with Golgi localization. The NIH-I329L cells were transfected with plasmid pDsRed-Monomer-Golgi to localize the protein to the Golgi (red, left) and then co-stained with a rat monoclonal high-affinity antibody against the HA, followed by a donkey affinity purified anti-rat IgG antibody conjugated with AMCA (left, middle). The colocalization is evident in the merged image (right panel). 2C ) The I329L protein was immunoprecipitated from lysates from the lentivirus-transduced NIH-I329L cell line with rabbit anti-HA antibodies bound to protein G beads. The bound protein was eluted and digested with EndoH (a) or PNGase (c) and examined by SDS-PAGE. Controls samples were treated with only EndoH buffer (b) or only PNGase buffer (d). 2D ) I329L protein is expressed at the cell surface. Biotinylated surface proteins from biotynylated NIH-I329L cells were collected with streptavidin beads, loaded onto a 10% SDS-PAGE gel and transferred to PVDF membranes, and I329L was revealed with a rat monoclonal antibody against HA conjugated with HRP (band in lane NIHI329L on the left). Lane NIHeGFP represents NIH-3T3 cells transduced with the empty lentivirus. Details are in Materials and methods

    Article Snippet: After rotating for 1 h at 4°C, the beads were removed by centrifugation, and the recombinant I329L was recovered from the supernatant by adding protein G beads (100 μl) and 5 μg of rabbit polyclonal affinity-purified anti-HA antibody (Sigma).

    Techniques: Transfection, Plasmid Preparation, Staining, Affinity Purification, Immunoprecipitation, SDS Page, Transduction

    Activated Smad proteins bind chromatin at the  Afp  distal promoter only when Foxa1 is bound and silent chromatin is altered. A–C, E , and  F , chromatin immunoprecipitation was performed on ES cells maintained in LIF or treated with RA for 4 days. DNA from immunoprecipitations for Foxa1 ( A ), Smad4 ( B ), P-Smad2 ( C ), H3 and H1 ( F ) was analyzed by real time PCR for binding to the  Afp  distal promoter and to the Nanog p53 response element. Results were graphed as percent bound minus IgG relative to input ( A-C ) or as normalized to LIF ( F ).  E , re-ChIP analysis reveals simultaneous association of Foxa1 and Smad4 at the  AFP  distal promoter in RA-treated ES cells, but not in LIF-treated ES cells. A 1° ChIP was done with Smad4 and reactions were pooled, released from protein A beads, and then reimmunoprecipitated for IgG or Foxa1.  Error bars  represent S.D. of at least three repetitions;  ns , not significant.  D , whole cell lysates from ES cells maintained with LIF or differentiated with RA were probed with antibodies to Smad4, Smad2, P-Smad2, and β-actin.  Triangles  indicate differential loading.

    Journal: The Journal of Biological Chemistry

    Article Title: Foxa1 Functions as a Pioneer Transcription Factor at Transposable Elements to Activate Afp during Differentiation of Embryonic Stem Cells *

    doi: 10.1074/jbc.M109.088096

    Figure Lengend Snippet: Activated Smad proteins bind chromatin at the Afp distal promoter only when Foxa1 is bound and silent chromatin is altered. A–C, E , and F , chromatin immunoprecipitation was performed on ES cells maintained in LIF or treated with RA for 4 days. DNA from immunoprecipitations for Foxa1 ( A ), Smad4 ( B ), P-Smad2 ( C ), H3 and H1 ( F ) was analyzed by real time PCR for binding to the Afp distal promoter and to the Nanog p53 response element. Results were graphed as percent bound minus IgG relative to input ( A-C ) or as normalized to LIF ( F ). E , re-ChIP analysis reveals simultaneous association of Foxa1 and Smad4 at the AFP distal promoter in RA-treated ES cells, but not in LIF-treated ES cells. A 1° ChIP was done with Smad4 and reactions were pooled, released from protein A beads, and then reimmunoprecipitated for IgG or Foxa1. Error bars represent S.D. of at least three repetitions; ns , not significant. D , whole cell lysates from ES cells maintained with LIF or differentiated with RA were probed with antibodies to Smad4, Smad2, P-Smad2, and β-actin. Triangles indicate differential loading.

    Article Snippet: The cell lysate was precleared with incubation with 20 μl of protein-A beads (50% slurry, Sigma) for 1 h. The precleared lysate was incubated overnight with antibodies: anti-Smad4 (DPC4, Upstate) and anti-rabbit IgG (Upstate).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    LGR5  and  PROM1  mRNAs interact with Lin28b protein (A) RNAs co-immunoprecipitating with Lin28b in colon cancer cells were reverse transcribed with an oligo-dT primer and qPCR performed for  GAPDH ,  IGF2 ,  LGR5 , and  PROM1  mRNAs. Fold change is relative to mRNA levels immunoprecipitated in the absence of Lin28b antibody, following normalization to  ACTB  (β-actin) mRNA levels. Error bars depict standard deviation from the mean. (B) Luciferase assays were performed to assess association of Lin28b protein with  LGR5  and  PROM1  3′ UTR sequences in colon cancer cells. Luciferase activity is enhanced by  LGR5  and  PROM1  3′ UTRs in the presence of increased  LIN28B  expression. Error bars depict standard deviation from mean.

    Journal: Oncogene

    Article Title: LIN28B fosters colon cancer migration, invasion, and transformation through let-7 dependent and independent mechanisms

    doi: 10.1038/onc.2011.131

    Figure Lengend Snippet: LGR5 and PROM1 mRNAs interact with Lin28b protein (A) RNAs co-immunoprecipitating with Lin28b in colon cancer cells were reverse transcribed with an oligo-dT primer and qPCR performed for GAPDH , IGF2 , LGR5 , and PROM1 mRNAs. Fold change is relative to mRNA levels immunoprecipitated in the absence of Lin28b antibody, following normalization to ACTB (β-actin) mRNA levels. Error bars depict standard deviation from the mean. (B) Luciferase assays were performed to assess association of Lin28b protein with LGR5 and PROM1 3′ UTR sequences in colon cancer cells. Luciferase activity is enhanced by LGR5 and PROM1 3′ UTRs in the presence of increased LIN28B expression. Error bars depict standard deviation from mean.

    Article Snippet: Protein A magnetic beads (Millipore, Billerica, MA) were washed in NT2 buffer (per ; ) and blocked in 5% BSA, 200 μg/ml yeast tRNA for 1 hour at 4°C.

    Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Standard Deviation, Luciferase, Activity Assay, Expressing

    Binding of biotinylated, full-length rgC1qR (MF) to immobilized protein A. Biotinylated rgC1qR (∼2 μg/ml) was incubated (60 min, 37°C) on protein A-coated microtiter wells. Binding was detected with AP-STRAV and pNPP substrate. C1q- and BSA-coated microtiter wells served as controls. Values represent means ± SD; n = 3. O.D., optical density.

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

    doi:

    Figure Lengend Snippet: Binding of biotinylated, full-length rgC1qR (MF) to immobilized protein A. Biotinylated rgC1qR (∼2 μg/ml) was incubated (60 min, 37°C) on protein A-coated microtiter wells. Binding was detected with AP-STRAV and pNPP substrate. C1q- and BSA-coated microtiter wells served as controls. Values represent means ± SD; n = 3. O.D., optical density.

    Article Snippet: The following chemicals and reagents were purchased from the sources indicated: protein A (Cowan I), human complement component C1q, bovine serum albumin (BSA), rabbit IgG, p -nitrophenyl phosphate (pNPP), S. aureus Cowan I and Wood 46 strains (formalin-treated cell suspensions), and dimethyl sulfoxide (DMSO), Sigma Chemical Co. (St. Louis, Mo.); alkaline phosphatase-conjugated goat anti-rabbit IgG, Organon Teknika Corp. (West Chester, Pa.); alkaline phosphatase-conjugated streptavidin (AP-STRAV), protein A-agarose, and sulfosuccinimidyl-6-biotinamidohexanoate (NHS-LC-biotin), Pierce Chemical Co. (Rockford, Ill.); Tween 20, J. T. Baker (Phillipsburg, N.J.); PD-10 columns, Pharmacia Biotech (Piscataway, N.J.).

    Techniques: Binding Assay, Incubation

    Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit IgG binding (series 1) is shown to demonstrate successful protein A hyperiodination. BSA-coated microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

    doi:

    Figure Lengend Snippet: Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit IgG binding (series 1) is shown to demonstrate successful protein A hyperiodination. BSA-coated microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.

    Article Snippet: The following chemicals and reagents were purchased from the sources indicated: protein A (Cowan I), human complement component C1q, bovine serum albumin (BSA), rabbit IgG, p -nitrophenyl phosphate (pNPP), S. aureus Cowan I and Wood 46 strains (formalin-treated cell suspensions), and dimethyl sulfoxide (DMSO), Sigma Chemical Co. (St. Louis, Mo.); alkaline phosphatase-conjugated goat anti-rabbit IgG, Organon Teknika Corp. (West Chester, Pa.); alkaline phosphatase-conjugated streptavidin (AP-STRAV), protein A-agarose, and sulfosuccinimidyl-6-biotinamidohexanoate (NHS-LC-biotin), Pierce Chemical Co. (Rockford, Ill.); Tween 20, J. T. Baker (Phillipsburg, N.J.); PD-10 columns, Pharmacia Biotech (Piscataway, N.J.).

    Techniques: Binding Assay, Ligand Binding Assay

    Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit IgG binding (series 1) is shown to demonstrate successful protein A hyperiodination. BSA-coated microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.

    Journal: Infection and Immunity

    Article Title: Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

    doi:

    Figure Lengend Snippet: Comparison of biotinylated rgC1qR (MF) (series 2) and rgC1qR (TF) (series 3) binding to microtiter well-immobilized staphylococcal protein A (Protein A) and hyperiodinated protein A (I-Protein A). Biotinylated ligand binding was quantified after 1 h at 37°C using AP-STRAV and pNPP substrate. Biotinylated rabbit IgG binding (series 1) is shown to demonstrate successful protein A hyperiodination. BSA-coated microtiter wells were used to determine background reactivity. Values represent means ± SD; n = 3. O.D., optical density.

    Article Snippet: The following chemicals and reagents were purchased from the sources indicated: protein A (Cowan I), human complement component C1q, bovine serum albumin (BSA), rabbit IgG, p -nitrophenyl phosphate (pNPP), S. aureus Cowan I and Wood 46 strains (formalin-treated cell suspensions), and dimethyl sulfoxide (DMSO), Sigma Chemical Co. (St. Louis, Mo.); alkaline phosphatase-conjugated goat anti-rabbit IgG, Organon Teknika Corp. (West Chester, Pa.); alkaline phosphatase-conjugated streptavidin (AP-STRAV), protein A-agarose, and sulfosuccinimidyl-6-biotinamidohexanoate (NHS-LC-biotin), Pierce Chemical Co. (Rockford, Ill.); Tween 20, J. T. Baker (Phillipsburg, N.J.); PD-10 columns, Pharmacia Biotech (Piscataway, N.J.).

    Techniques: Binding Assay, Ligand Binding Assay

    Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: Laminin; lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p

    Journal: Acta Neuropathologica Communications

    Article Title: Lack of robust satellite cell activation and muscle regeneration during the progression of Pompe disease

    doi: 10.1186/s40478-015-0243-x

    Figure Lengend Snippet: Satellite cell numbers remain unchanged and are independent of Pompe disease severity. a Identification of satellite cells (white arrows) by immunofluorescent analysis of Pax7 in human skeletal muscle biopsies. Upper left: Pax7; upper right: Hoechst; lower left: Laminin; lower right: merge. b , c Quantification of Pax7-positive cells in all Pompe biopsies examined in this study. Healthy control biopsies are included, as well as biopsies from two DMD patients. b Number of satellite cells (SCs)/mm 2 . c Percentage of SCs/nuclei. d Representative examples of each Pompe patient group, age-matched controls, and a DMD patient. Pompe patients: group 1 n = 3, group 2 n = 4, group 3 n = 4, group 4 n = 3. DMD patients: n = 2. Controls: infants n = 2, juveniles n = 2 and adult n = 5. For both Fig. 2b and c: Group 1 Pompe patients were statistically significant from other Pompe patient groups ( p

    Article Snippet: Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400).

    Techniques:

    Impaired muscle regeneration in Pompe patients. Immunofluorescent analysis of embryonic myosin heavy chain (eMyHC in red) expression in to detect actively regenerating myofibers. Muscle sections were co-stained for Laminin ( in green ) to visualize the fiber outline, nuclei were stained with Hoechst ( in blue ). Representative examples are shown. Two examples from classic infantile Pompe patients and DMD patients are derived from different patients

    Journal: Acta Neuropathologica Communications

    Article Title: Lack of robust satellite cell activation and muscle regeneration during the progression of Pompe disease

    doi: 10.1186/s40478-015-0243-x

    Figure Lengend Snippet: Impaired muscle regeneration in Pompe patients. Immunofluorescent analysis of embryonic myosin heavy chain (eMyHC in red) expression in to detect actively regenerating myofibers. Muscle sections were co-stained for Laminin ( in green ) to visualize the fiber outline, nuclei were stained with Hoechst ( in blue ). Representative examples are shown. Two examples from classic infantile Pompe patients and DMD patients are derived from different patients

    Article Snippet: Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400).

    Techniques: Expressing, Staining, Derivative Assay