dynabeads - protein Thermo Fisher Search Results


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  • 90
    Thermo Fisher dynabeads myone
    Dynabeads Myone, supplied by Thermo Fisher, 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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    Thermo Fisher dynabeads protein g
    COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with <t>Dynabeads</t> <t>Protein</t> G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.
    Dynabeads Protein G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 15450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads protein a
    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using <t>Dynabeads</t> <t>Protein</t> A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004
    Dynabeads Protein A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tosylactivated myone dynabeads
    Identification of CD4-binding site antibodies. ( A ) ELISA with RSC3 and RSC3∆371I/P363N recombinant proteins. 12 BCN plasma samples were tested at a dilution of 1:100 to screen for the presence of CD4 binding site directed antibodies. Four of the 12 BCN plasma samples tested demonstrated significantly stronger binding to the wild type RSC3 protein as compared to the mutant protein. ( B ) RSC3 and RSC3∆371I/P363N specific antibodies were eluted from four BCN plasma samples which revealed the presence of CD4 binding site targeting antibodies (NAB033, NAB059, NAB063 and NAB069) using tosyl activated <t>MyOne</t> <t>Dynabeads.</t> ELISA was again performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) with RSC3 and RSC3∆371I/P363N recombinant proteins at 2 μg/ml. Monoclonal antibodies VRC01, VRC03, B12 and 3BNC117 were used as positive controls and HHP as negative control. ( C ) Neutralization titer (IC 50 ) assay performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) against four pseudoviruses and one mutant (JR-FL D279A). VRC01 and b12 MAbs were used as positive control bNabs against CD4BS and MuLV, as negative control.
    Tosylactivated Myone Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher invitrogen dynabeads m 270
    Paramagnetic bead manipulation of 1.0 mg/mL solution of <t>Invitrogen</t> Dynabeads M-270 2.8 μm with four magnet positions and the resulting bead droplet. The left-hand droplet in each image was dispensed prior to application of the magnetic field. Approximate locations of the field wells are circled with a dotted line.
    Invitrogen Dynabeads M 270, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads co immunoprecipitation kit
    G0S2 binds to ATGL in the liver. Mice were injected via the tail vein with an Ad-G0S2 vector and killed 4 days later. (A) Immunofluorescent analysis of liver sections with the anti-G0S2 and anti-ATGL antibodies. Bound primary antibodies were visualized, respectively, with FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG. The sections were visualized under laser confocal microscopy. LDs in the livers were visualized under a phase-contrast microscope. The arrows indicate positive staining. Scale bar = 10 µm. (B) Anti-G0S2 and anti-ATGL western blot (WB) analyses were performed on ATGL or mouse monoclonal IgG immunoprecipitates (IP) prepared with the anti-ATGL antibody or IgG affinity <t>Dynabeads</t> (left panel). To control for equal loading, equal amounts by volume of crude extract for the <t>immunoprecipitation</t> experiments were loaded onto the SDS-PAGE gel for immunoblotting (right panel).
    Dynabeads Co Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads m 270 epoxy beads
    Size distribution analysis and differences in β3 integrin levels in ExVs from plasma of prostate cancer patients compared to subjects not affected by cancer. (A) NTA of ExVs isolated by differential ultracentrifugation from plasma of individuals not affected by cancer (left panel) and prostate cancer patients (right panel). (B) IB analysis of β3, CD9, CD81, and αv levels in ExVs isolated by differential ultracentrifugation from plasma of prostate cancer patients compared to age-matched individuals not affected by cancer. CANX was analyzed as a marker absent in exosomes. Lanes 1, 2, and 3: EV lysates isolated after the plasma was pooled from at least two subjects not affected by cancer (total of 7 biological samples represented in 3 lanes); lanes 4–8: EV lysates from individual patients. 30 μg of exosome lysates were loaded in each lane. (C) lodixanol gradient purified Exosomes (Exo) from prostate cancer patient plasma (pooled from  n  = 3) were immunocaptured with an antibody to Prostate Specific Membrane Antigen (PSMA) or isotype rabbit immunoglobulin (Rb-IgG) conjugated with Dynabeads M-270 epoxy magnetic beads, according to the manufacturer’s protocol. The immunocaptured whole exosomes were lysed with RIPA buffer, and lysates were separated by SDS-PAGE (7.5% gel). IB analysis shows expression of β3, CD9 and CD63 (exosomal markers), Trop-2, and PSMA; in contrast, TSG101 (exosomal marker), CANX and EpCAM were not detected. HC-IgG, heavy chain IgG.
    Dynabeads M 270 Epoxy Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher immunoprecipitation kit
    HMGB1 from ischemic myocardium and RAGE receptors on splenocytes mediate the IS exacerbating effects of 40-IHH A: Western blot analysis showing that 40-IHH had significantly higher HMGB1 levels in heart homogenates than in sham or 10-IHH. B: <t>Immunoprecipitation</t> was used to deplete HMGB1 in heart homogenates, which was confirmed by Western blots. C: Depletion of HMGB1 in 40-IHH completely abrogated its IS exacerbating effect. D: 40-IHH completely failed to increase IS in either RAGE −/− mice or splenectomized WT mice with adoptive transfer of RAGE −/− splenocytes . N = ≥5 mice/group. IP: immunoprecipitation.
    Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads protein g immunoprecipitation kit
    Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with <t>Dynabeads/anti–enhanced</t> green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, <t>immunoprecipitation.</t>
    Dynabeads Protein G Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher g dynabeads
    Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with <t>Dynabeads/anti–enhanced</t> green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, <t>immunoprecipitation.</t>
    G Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads antibody coupling kit
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
    Dynabeads Antibody Coupling Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fresh dynabead protein g
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
    Fresh Dynabead Protein G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher magnet starter
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
    Magnet Starter, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynal dynabeads protein g
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
    Dynal Dynabeads Protein G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protein a conjugated dynabeads 280
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
    Protein A Conjugated Dynabeads 280, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher coated dynabeads
    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
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    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated <t>dynabeads.</t> For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.
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    Depletion of NEDD4 compromises assembly of PRC2-Ezh1 complex and H3K27me3 occupancy on mCK, MyoG and MYH8 genomic loci under oxidative stress conditions. a , b Interaction between SUZ12 and EED was determined in scramble and NEDD4 knockdown stable cell lines under normal and oxidative stress conditions. Nuclear extracts from scramble and NEDD4 KD cell lines under normal and stress conditions were immunoprecipitated with SUZ12 antibody, associated protein complexes were eluted with 2XLDS loading buffer. SUZ12 and EED were detected using anti-SUZ12 and anti-EED. Protein A <t>Dynabeads</t> alone were incubated with nuclear extracts and used as mock control. Ponceau S staining was used as loading control. c , d ChIP-qPCR analysis of Ezh1α occupancy and H3K27me3 status on genomic loci of mCK enhancer, MyoG promoter, MYH8 and Neurog1. Chromatin immunoprecipitation (ChIP) was performed using chromatin from scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions against Ezh1α or H3K27me3 antibody. Precipitated DNA were measured using qPCR assay with specific primers corresponding to mCK enhancer, MyoG promoter, MYH8 and NeuroG1 genomic regions. ChIP enrichments are shown as percentage of input. Data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t -test p value. e Transcription level of NEDD4, mCK, MyoG, MYH8 and Atrogin1 were analyzed using RT-qPCR in scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions. Data were expressed as mean ± SD from three biological replicates. GAPDH was normalized to get relative expression of each target. Values above each bar indicate Student’s t -test p value
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    Depletion of NEDD4 compromises assembly of PRC2-Ezh1 complex and H3K27me3 occupancy on mCK, MyoG and MYH8 genomic loci under oxidative stress conditions. a , b Interaction between SUZ12 and EED was determined in scramble and NEDD4 knockdown stable cell lines under normal and oxidative stress conditions. Nuclear extracts from scramble and NEDD4 KD cell lines under normal and stress conditions were immunoprecipitated with SUZ12 antibody, associated protein complexes were eluted with 2XLDS loading buffer. SUZ12 and EED were detected using anti-SUZ12 and anti-EED. Protein A <t>Dynabeads</t> alone were incubated with nuclear extracts and used as mock control. Ponceau S staining was used as loading control. c , d ChIP-qPCR analysis of Ezh1α occupancy and H3K27me3 status on genomic loci of mCK enhancer, MyoG promoter, MYH8 and Neurog1. Chromatin immunoprecipitation (ChIP) was performed using chromatin from scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions against Ezh1α or H3K27me3 antibody. Precipitated DNA were measured using qPCR assay with specific primers corresponding to mCK enhancer, MyoG promoter, MYH8 and NeuroG1 genomic regions. ChIP enrichments are shown as percentage of input. Data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t -test p value. e Transcription level of NEDD4, mCK, MyoG, MYH8 and Atrogin1 were analyzed using RT-qPCR in scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions. Data were expressed as mean ± SD from three biological replicates. GAPDH was normalized to get relative expression of each target. Values above each bar indicate Student’s t -test p value
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    CLC-3 is phosphorylated by Ca 2+ /calmodulin kinase II (CaMKII) tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A , tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B , α-CLC-3 was incubated with protein A-conjugated magnetic <t>Dynabeads</t> and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C , CLC-3 is phosphorylated by CaMKII in vitro . Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.
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    CLC-3 is phosphorylated by Ca 2+ /calmodulin kinase II (CaMKII) tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A , tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B , α-CLC-3 was incubated with protein A-conjugated magnetic <t>Dynabeads</t> and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C , CLC-3 is phosphorylated by CaMKII in vitro . Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.
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    CLC-3 is phosphorylated by Ca 2+ /calmodulin kinase II (CaMKII) tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A , tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B , α-CLC-3 was incubated with protein A-conjugated magnetic <t>Dynabeads</t> and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C , CLC-3 is phosphorylated by CaMKII in vitro . Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.
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    Asn-43 mutants still interact with RIP2. Co-immunoprecipitation experiments were performed in HEK293 cells transfected with the constructs detailed in either the presence ( A ) or absence ( B ) of stimulation with 100 ng/ml iE-DAP. Complexes were immunoprecipitated using anti-FLAG antibody immobilized on Protein G-coated <t>Dynabeads</t> and analyzed with the antibodies detailed. Blots are representative of at least three separate experiments. co-purification of E. coli -expressed GST-NOD1 CARD and GB1-RIP2-CARD ( C ) or His-MBP-NOD1-CARD and GB1-RIP2-CARD ( D ). Expressed proteins were pooled during lysis, co-purified with glutathione-Sepharose or amylose resin as appropriate, and detected by Instant Blue (Expedeon) staining. In both C and D , T represents total lysate, and E represents eluted fraction. The position of recombinant proteins is marked. Images are representative of at least three independent experiments.
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    Asn-43 mutants still interact with RIP2. Co-immunoprecipitation experiments were performed in HEK293 cells transfected with the constructs detailed in either the presence ( A ) or absence ( B ) of stimulation with 100 ng/ml iE-DAP. Complexes were immunoprecipitated using anti-FLAG antibody immobilized on Protein G-coated <t>Dynabeads</t> and analyzed with the antibodies detailed. Blots are representative of at least three separate experiments. co-purification of E. coli -expressed GST-NOD1 CARD and GB1-RIP2-CARD ( C ) or His-MBP-NOD1-CARD and GB1-RIP2-CARD ( D ). Expressed proteins were pooled during lysis, co-purified with glutathione-Sepharose or amylose resin as appropriate, and detected by Instant Blue (Expedeon) staining. In both C and D , T represents total lysate, and E represents eluted fraction. The position of recombinant proteins is marked. Images are representative of at least three independent experiments.
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    Image Search Results


    COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis

    doi: 10.1073/pnas.1816268116

    Figure Lengend Snippet: COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.

    Article Snippet: HEK293T cells were transiently transfected with 10 μg of the respective combinations of constructs for 24 h. Ubiquitinated RUP2-HA was detected by IP with 2 μL of anti-HA (Sigma–Aldrich) coupled with 10 μL of Dynabeads Protein G (Thermo Fisher Scientific) at 4 °C for 5 h. The pellets were washed three times and eluted with 2× SDS protein loading buffer.

    Techniques: Negative Control, In Vitro, Purification, Incubation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Magnetic Beads

    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Expressing, Fluorescence, Plasmid Preparation, Staining, Negative Control

    Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Negative Control, Western Blot, Purification, Positive Control, SDS Page, Staining

    Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Journal: eLife

    Article Title: FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory

    doi: 10.7554/eLife.46919

    Figure Lengend Snippet: Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Article Snippet: The remaining lysates was precleared by incubating with 50 μl Protein A Dynabeads (Invitrogen) for 45 min at 4°C with rotation then incubated with 20 μg anti-HA (ab9110, abcam) for 2 hr at 4°C with rotation.

    Techniques: Immunoprecipitation, Titration, Incubation, Protein Extraction, Isolation, Protein Concentration, Western Blot, Concentration Assay

    PAI1 inhibits binding of EA-tPA to immunopurified NMDA-R. (A) Extracts of mouse cortex were incubated with NMDA-R NR1 subunit-specific antibody (NR1) or with NS-IgG (IgG) coupled to Protein-G–Dynabeads. Bound proteins were immunoprecipitated in

    Journal: Journal of Cell Science

    Article Title: PAI1 blocks NMDA receptor-mediated effects of tissue-type plasminogen activator on cell signaling and physiology

    doi: 10.1242/jcs.217083

    Figure Lengend Snippet: PAI1 inhibits binding of EA-tPA to immunopurified NMDA-R. (A) Extracts of mouse cortex were incubated with NMDA-R NR1 subunit-specific antibody (NR1) or with NS-IgG (IgG) coupled to Protein-G–Dynabeads. Bound proteins were immunoprecipitated in

    Article Snippet: Extracts were cleared by centrifugation at 13,000 g for 10 min, diluted five-fold in immunoprecipitation bufffer and then pre-cleared by incubation with Protein G–Dynabeads (Thermo Scientific) in the absence of antibodies for 2 h at 4°C.

    Techniques: Binding Assay, Incubation, Immunoprecipitation

    Immunoisolation of CHT-containing vesicles. A , Compared with non-antibody-coupled protein A Dynabeads, VAChT polyclonal antiserum-coated beads efficiently isolate vesicles containing CHT and VAChT from the presynaptic vesicle-enriched striatal LS1 preparation. In the reciprocal experiment the antiserum directed against the cytoplasmic C terminus of CHT also effectively captures both CHT and a large fraction of the VAChT immunoreactivity. None of these assays captures appreciable quantities of the general synaptic vesicle marker synaptotagmin I or the dopaminergic neuron-specific dopamine transporter (DAT). B , Immunodepletion analysis of the synaptic vesicle-containing fractions from glycerol velocity gradients demonstrates that, when the CHT polyclonal antiserum clears the majority of CHT-containing vesicles (91±2%) from this fraction, VAChT immunoreactivity representing cholinergic synaptic vesicles also is depleted by one-half (45±5%). The depletion is specific for cholinergic synaptic vesicles because VMAT2 levels are unaltered (4± 6% depletion) between the control beads and the CHT antibody-coated beads ( n = 4). C , In reciprocal experiments using the VAChT antiserum for the immunodepletion of cholinergic synaptic vesicles, depletion of VAChT immunoreactivity by 88 ± 2% is accompanied by a proportional and near-complete (89 ± 2%) depletion of CHT-containing vesicles. Immunoblotting for VMAT2 demonstrates the selective immunodepletion of cholinergic synaptic vesicles ( n = 3). D , The monoclonal antibody raised against the C terminus of CHT also isolates CHT-containing vesicles that are positive for VAChT. Immunoisolation of synaptic vesicles with a synaptophysin antibody (clone 7.2) also results in the purification of CHT- and VAChT-containing vesicles. Notably, VMAT2 immunoreactivity is absent from the nonspecific mouse IgG-coated control protein G Sepharose beads and the CHT antibody beads but is present on the synaptophysin antibody beads. E , Closer examination of CHT monoclonal antibody beads with longer film exposures reveals the enrichment for CHT, VAChT, synaptophysin, and Rab3A as compared with nonspecific mouse IgG beads. Equivalent amounts of IgG heavy chain are present in each lane. F , Immunoisolation of Rab3A-positive vesicles depletes striatal LS1 fractions of CHT, Rab3A, and synaptophysin, but not DAT or GAT1 (results representative of 3 independent experiments). G , CHT-containing vesicles exhibit vesamicol-sensitive ACh uptake. After the loading of striatal synaptosomes with [ 3 H]-choline ± vesamicol, CHT-specific antiserum is effective at immunoisolating vesamicol-sensitive [ 3 H]-ACh-containing vesicles (2.8 ± 0.4 vs 1.1 ± 0.2 fmol of ACh; n = 3) but is no better than the preimmune serum with respect to the isolation of vesicles containing reserpine-sensitive dopamine (DA) counts (14.1 ± 3.7 vs 14.2 ± 2.6 fmol DA for anti-CHT serum and preimmune serum, respectively). Averaged data are presented from three separate experiments performed in duplicate ± SEM; * p

    Journal: The Journal of Neuroscience

    Article Title: Vesicular Localization and Activity-Dependent Trafficking of Presynaptic Choline Transporters

    doi: 10.1523/JNEUROSCI.23-30-09697.2003

    Figure Lengend Snippet: Immunoisolation of CHT-containing vesicles. A , Compared with non-antibody-coupled protein A Dynabeads, VAChT polyclonal antiserum-coated beads efficiently isolate vesicles containing CHT and VAChT from the presynaptic vesicle-enriched striatal LS1 preparation. In the reciprocal experiment the antiserum directed against the cytoplasmic C terminus of CHT also effectively captures both CHT and a large fraction of the VAChT immunoreactivity. None of these assays captures appreciable quantities of the general synaptic vesicle marker synaptotagmin I or the dopaminergic neuron-specific dopamine transporter (DAT). B , Immunodepletion analysis of the synaptic vesicle-containing fractions from glycerol velocity gradients demonstrates that, when the CHT polyclonal antiserum clears the majority of CHT-containing vesicles (91±2%) from this fraction, VAChT immunoreactivity representing cholinergic synaptic vesicles also is depleted by one-half (45±5%). The depletion is specific for cholinergic synaptic vesicles because VMAT2 levels are unaltered (4± 6% depletion) between the control beads and the CHT antibody-coated beads ( n = 4). C , In reciprocal experiments using the VAChT antiserum for the immunodepletion of cholinergic synaptic vesicles, depletion of VAChT immunoreactivity by 88 ± 2% is accompanied by a proportional and near-complete (89 ± 2%) depletion of CHT-containing vesicles. Immunoblotting for VMAT2 demonstrates the selective immunodepletion of cholinergic synaptic vesicles ( n = 3). D , The monoclonal antibody raised against the C terminus of CHT also isolates CHT-containing vesicles that are positive for VAChT. Immunoisolation of synaptic vesicles with a synaptophysin antibody (clone 7.2) also results in the purification of CHT- and VAChT-containing vesicles. Notably, VMAT2 immunoreactivity is absent from the nonspecific mouse IgG-coated control protein G Sepharose beads and the CHT antibody beads but is present on the synaptophysin antibody beads. E , Closer examination of CHT monoclonal antibody beads with longer film exposures reveals the enrichment for CHT, VAChT, synaptophysin, and Rab3A as compared with nonspecific mouse IgG beads. Equivalent amounts of IgG heavy chain are present in each lane. F , Immunoisolation of Rab3A-positive vesicles depletes striatal LS1 fractions of CHT, Rab3A, and synaptophysin, but not DAT or GAT1 (results representative of 3 independent experiments). G , CHT-containing vesicles exhibit vesamicol-sensitive ACh uptake. After the loading of striatal synaptosomes with [ 3 H]-choline ± vesamicol, CHT-specific antiserum is effective at immunoisolating vesamicol-sensitive [ 3 H]-ACh-containing vesicles (2.8 ± 0.4 vs 1.1 ± 0.2 fmol of ACh; n = 3) but is no better than the preimmune serum with respect to the isolation of vesicles containing reserpine-sensitive dopamine (DA) counts (14.1 ± 3.7 vs 14.2 ± 2.6 fmol DA for anti-CHT serum and preimmune serum, respectively). Averaged data are presented from three separate experiments performed in duplicate ± SEM; * p

    Article Snippet: For immunoisolation of CHT-containing vesicles rabbit antisera against CHT (Ab21), VAChT or Rab3A was coupled to protein A-Sepharose beads (Amersham Biosciences) or to either sheep anti-rabbit IgG-coated or protein A-coupled paramagnetic Dynabeads (Dynal, Lake Success, NY) in PBS and 1 m m EDTA, pH 7.4, for at least 4 hr at 4°C.

    Techniques: Marker, Purification, Isolation

    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Journal: eLife

    Article Title: Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis

    doi: 10.7554/eLife.00675

    Figure Lengend Snippet: EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Article Snippet: We then substituted Dynabeads Protein A (Invitrogen, cat#100-1D) and Dynabeads M-280 Sheep anti-Rabbit IgG (Invitrogen, cat#112-04D) for the salmon sperm DNA blocked Protein A agarose beads recommended in the protocol (4), as to avoid sequencing of salmon sperm DNA.

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

    Identification of CD4-binding site antibodies. ( A ) ELISA with RSC3 and RSC3∆371I/P363N recombinant proteins. 12 BCN plasma samples were tested at a dilution of 1:100 to screen for the presence of CD4 binding site directed antibodies. Four of the 12 BCN plasma samples tested demonstrated significantly stronger binding to the wild type RSC3 protein as compared to the mutant protein. ( B ) RSC3 and RSC3∆371I/P363N specific antibodies were eluted from four BCN plasma samples which revealed the presence of CD4 binding site targeting antibodies (NAB033, NAB059, NAB063 and NAB069) using tosyl activated MyOne Dynabeads. ELISA was again performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) with RSC3 and RSC3∆371I/P363N recombinant proteins at 2 μg/ml. Monoclonal antibodies VRC01, VRC03, B12 and 3BNC117 were used as positive controls and HHP as negative control. ( C ) Neutralization titer (IC 50 ) assay performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) against four pseudoviruses and one mutant (JR-FL D279A). VRC01 and b12 MAbs were used as positive control bNabs against CD4BS and MuLV, as negative control.

    Journal: Scientific Reports

    Article Title: Broad and potent cross clade neutralizing antibodies with multiple specificities in the plasma of HIV-1 subtype C infected individuals

    doi: 10.1038/srep46557

    Figure Lengend Snippet: Identification of CD4-binding site antibodies. ( A ) ELISA with RSC3 and RSC3∆371I/P363N recombinant proteins. 12 BCN plasma samples were tested at a dilution of 1:100 to screen for the presence of CD4 binding site directed antibodies. Four of the 12 BCN plasma samples tested demonstrated significantly stronger binding to the wild type RSC3 protein as compared to the mutant protein. ( B ) RSC3 and RSC3∆371I/P363N specific antibodies were eluted from four BCN plasma samples which revealed the presence of CD4 binding site targeting antibodies (NAB033, NAB059, NAB063 and NAB069) using tosyl activated MyOne Dynabeads. ELISA was again performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) with RSC3 and RSC3∆371I/P363N recombinant proteins at 2 μg/ml. Monoclonal antibodies VRC01, VRC03, B12 and 3BNC117 were used as positive controls and HHP as negative control. ( C ) Neutralization titer (IC 50 ) assay performed with eluted IgG antibody (concentration 10 to 0.0001 μg/ml) against four pseudoviruses and one mutant (JR-FL D279A). VRC01 and b12 MAbs were used as positive control bNabs against CD4BS and MuLV, as negative control.

    Article Snippet: Protein-paramagnet bead coupling, adsorption and elution of RSC3- specific antibodies Paramagnetic polystyrene, Tosylactivated MyOne Dynabeads (Life Technologies) were coupled with recombinant HIV-1 RSC3 and double mutant RSC3Δ371I/P363N proteins or pIndie MPER peptide as per the manufacturer’s instructions.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Mutagenesis, Concentration Assay, Negative Control, Neutralization, Positive Control

    Paramagnetic bead manipulation of 1.0 mg/mL solution of Invitrogen Dynabeads M-270 2.8 μm with four magnet positions and the resulting bead droplet. The left-hand droplet in each image was dispensed prior to application of the magnetic field. Approximate locations of the field wells are circled with a dotted line.

    Journal: Biotechnology journal

    Article Title: Picoliter DNA Sequencing Chemistry on an Electrowetting-based Digital Microfluidic Platform

    doi: 10.1002/biot.201000324

    Figure Lengend Snippet: Paramagnetic bead manipulation of 1.0 mg/mL solution of Invitrogen Dynabeads M-270 2.8 μm with four magnet positions and the resulting bead droplet. The left-hand droplet in each image was dispensed prior to application of the magnetic field. Approximate locations of the field wells are circled with a dotted line.

    Article Snippet: The beads used in these tests were Polysciences Fluoresbrite 2 μm and Polybead 10 μm carboxyl-functionalized polystyrene spheres and Invitrogen Dynabeads M-270 2.8 μm streptavidin-functionalized paramagnetic beads.

    Techniques:

    G0S2 binds to ATGL in the liver. Mice were injected via the tail vein with an Ad-G0S2 vector and killed 4 days later. (A) Immunofluorescent analysis of liver sections with the anti-G0S2 and anti-ATGL antibodies. Bound primary antibodies were visualized, respectively, with FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG. The sections were visualized under laser confocal microscopy. LDs in the livers were visualized under a phase-contrast microscope. The arrows indicate positive staining. Scale bar = 10 µm. (B) Anti-G0S2 and anti-ATGL western blot (WB) analyses were performed on ATGL or mouse monoclonal IgG immunoprecipitates (IP) prepared with the anti-ATGL antibody or IgG affinity Dynabeads (left panel). To control for equal loading, equal amounts by volume of crude extract for the immunoprecipitation experiments were loaded onto the SDS-PAGE gel for immunoblotting (right panel).

    Journal: PLoS ONE

    Article Title: The G0/G1 Switch Gene 2 Is an Important Regulator of Hepatic Triglyceride Metabolism

    doi: 10.1371/journal.pone.0072315

    Figure Lengend Snippet: G0S2 binds to ATGL in the liver. Mice were injected via the tail vein with an Ad-G0S2 vector and killed 4 days later. (A) Immunofluorescent analysis of liver sections with the anti-G0S2 and anti-ATGL antibodies. Bound primary antibodies were visualized, respectively, with FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG. The sections were visualized under laser confocal microscopy. LDs in the livers were visualized under a phase-contrast microscope. The arrows indicate positive staining. Scale bar = 10 µm. (B) Anti-G0S2 and anti-ATGL western blot (WB) analyses were performed on ATGL or mouse monoclonal IgG immunoprecipitates (IP) prepared with the anti-ATGL antibody or IgG affinity Dynabeads (left panel). To control for equal loading, equal amounts by volume of crude extract for the immunoprecipitation experiments were loaded onto the SDS-PAGE gel for immunoblotting (right panel).

    Article Snippet: Co-immunoprecipitation and western blot analyses For the co-immunoprecipitation assays, 300 µg of total protein extract from mouse livers were immunoprecipitated with the anti-ATGL antibody or pre-immune IgG by using a Dynabeads® Co-Immunoprecipitation Kit (Invitrogen, CA, USA) and then subjected to western blot analysis with the anti-G0S2 antibody.

    Techniques: Mouse Assay, Injection, Plasmid Preparation, Confocal Microscopy, Microscopy, Staining, Western Blot, Immunoprecipitation, SDS Page

    Size distribution analysis and differences in β3 integrin levels in ExVs from plasma of prostate cancer patients compared to subjects not affected by cancer. (A) NTA of ExVs isolated by differential ultracentrifugation from plasma of individuals not affected by cancer (left panel) and prostate cancer patients (right panel). (B) IB analysis of β3, CD9, CD81, and αv levels in ExVs isolated by differential ultracentrifugation from plasma of prostate cancer patients compared to age-matched individuals not affected by cancer. CANX was analyzed as a marker absent in exosomes. Lanes 1, 2, and 3: EV lysates isolated after the plasma was pooled from at least two subjects not affected by cancer (total of 7 biological samples represented in 3 lanes); lanes 4–8: EV lysates from individual patients. 30 μg of exosome lysates were loaded in each lane. (C) lodixanol gradient purified Exosomes (Exo) from prostate cancer patient plasma (pooled from  n  = 3) were immunocaptured with an antibody to Prostate Specific Membrane Antigen (PSMA) or isotype rabbit immunoglobulin (Rb-IgG) conjugated with Dynabeads M-270 epoxy magnetic beads, according to the manufacturer’s protocol. The immunocaptured whole exosomes were lysed with RIPA buffer, and lysates were separated by SDS-PAGE (7.5% gel). IB analysis shows expression of β3, CD9 and CD63 (exosomal markers), Trop-2, and PSMA; in contrast, TSG101 (exosomal marker), CANX and EpCAM were not detected. HC-IgG, heavy chain IgG.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

    doi: 10.1016/j.matbio.2018.08.004

    Figure Lengend Snippet: Size distribution analysis and differences in β3 integrin levels in ExVs from plasma of prostate cancer patients compared to subjects not affected by cancer. (A) NTA of ExVs isolated by differential ultracentrifugation from plasma of individuals not affected by cancer (left panel) and prostate cancer patients (right panel). (B) IB analysis of β3, CD9, CD81, and αv levels in ExVs isolated by differential ultracentrifugation from plasma of prostate cancer patients compared to age-matched individuals not affected by cancer. CANX was analyzed as a marker absent in exosomes. Lanes 1, 2, and 3: EV lysates isolated after the plasma was pooled from at least two subjects not affected by cancer (total of 7 biological samples represented in 3 lanes); lanes 4–8: EV lysates from individual patients. 30 μg of exosome lysates were loaded in each lane. (C) lodixanol gradient purified Exosomes (Exo) from prostate cancer patient plasma (pooled from n = 3) were immunocaptured with an antibody to Prostate Specific Membrane Antigen (PSMA) or isotype rabbit immunoglobulin (Rb-IgG) conjugated with Dynabeads M-270 epoxy magnetic beads, according to the manufacturer’s protocol. The immunocaptured whole exosomes were lysed with RIPA buffer, and lysates were separated by SDS-PAGE (7.5% gel). IB analysis shows expression of β3, CD9 and CD63 (exosomal markers), Trop-2, and PSMA; in contrast, TSG101 (exosomal marker), CANX and EpCAM were not detected. HC-IgG, heavy chain IgG.

    Article Snippet: Rabbit monoclonal antibody to PSMA (ab-133,579, Abcam; 100 μg) or isotype rabbit immunoglobulin (Rb-IgG, 100 μg), each was conjugated with 5 mg of Dynabeads™ M-270 Epoxy beads (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Isolation, Marker, Purification, Magnetic Beads, SDS Page, Expressing

    HMGB1 from ischemic myocardium and RAGE receptors on splenocytes mediate the IS exacerbating effects of 40-IHH A: Western blot analysis showing that 40-IHH had significantly higher HMGB1 levels in heart homogenates than in sham or 10-IHH. B: Immunoprecipitation was used to deplete HMGB1 in heart homogenates, which was confirmed by Western blots. C: Depletion of HMGB1 in 40-IHH completely abrogated its IS exacerbating effect. D: 40-IHH completely failed to increase IS in either RAGE −/− mice or splenectomized WT mice with adoptive transfer of RAGE −/− splenocytes . N = ≥5 mice/group. IP: immunoprecipitation.

    Journal: Basic research in cardiology

    Article Title: The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE

    doi: 10.1007/s00395-016-0583-0

    Figure Lengend Snippet: HMGB1 from ischemic myocardium and RAGE receptors on splenocytes mediate the IS exacerbating effects of 40-IHH A: Western blot analysis showing that 40-IHH had significantly higher HMGB1 levels in heart homogenates than in sham or 10-IHH. B: Immunoprecipitation was used to deplete HMGB1 in heart homogenates, which was confirmed by Western blots. C: Depletion of HMGB1 in 40-IHH completely abrogated its IS exacerbating effect. D: 40-IHH completely failed to increase IS in either RAGE −/− mice or splenectomized WT mice with adoptive transfer of RAGE −/− splenocytes . N = ≥5 mice/group. IP: immunoprecipitation.

    Article Snippet: Depletion of HMGB1 protein in IHH was achieved by immunoprecipitation with a Dynabeads® Protein A Immunoprecipitation Kit according to the manufacturer’s instructions (Life Technologies, Grand Island, NY).

    Techniques: Western Blot, Immunoprecipitation, Mouse Assay, Adoptive Transfer Assay

    Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with Dynabeads/anti–enhanced green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, immunoprecipitation.

    Journal: The Journal of Infectious Diseases

    Article Title: Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells

    doi: 10.1093/infdis/jiy294

    Figure Lengend Snippet: Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with Dynabeads/anti–enhanced green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, immunoprecipitation.

    Article Snippet: Proteins were precipitated using a rabbit anti-eGFP antibody and Dynabeads protein G immunoprecipitation kit (Thermo Fisher) according to the manufacturer’s protocol.

    Techniques: Transfection, Staining, Incubation, Expressing, Immunoprecipitation

    Expression of the diNAcBac glycan in V. cholerae results in multiple glycosylated proteins in a PglL Vc -dependent manner. (A) Shown is a representative immunoblot using the anti-diNAcBac antibody (npg1) to detect diNAcBac-glycosylated proteins and the corresponding Kang-stained SDS-gel of whole cell extracts derived from Δ pglL Vc Δ rbmD with or without in trans expression of respective O -OTases PglL Vc or RbmD (+ = pMMBneo-pglL Vc or pMMBneo-rbmD; – = pMMBneo) as well as the diNAcBac glycan (+ = pACYCpglFBCD; – = pACYC184). Only the presence of PglL Vc and diNAcBAc yielded in the detection of multiple bands. (B) Shown is an immunoblot using the npg1-antibody to detect diNAcBac-glycosylated proteins and the corresponding Kang-stained SDS-gel of immunopreciptated samples of Δ pglL Vc Δ rbmD expressing the diNAcBac glycan (pACYCpglFBCD) in the presence (+ = pMMBneo-pglL Vc ) or absence (- = pMMBneo) of PglL Vc . Immunoprecipitation was performed using npg1 immobilized onto Dynabeads coupled with protein G in combination with membrane and periplasmic enriched protein samples from the respective strains. Selected protein bands, which appear to be enriched upon presence of PglL Vc on the SDS-gel and resulted in a decent signal on the immunoblot, were excised and subjected to MS. Identified proteins are indicated on the right with their respective position on the gel, protein identities and accession numbers. (A,B) Lines to the left indicate the molecular masses of the protein standards in kDa.

    Journal: Frontiers in Microbiology

    Article Title: A Broad Spectrum Protein Glycosylation System Influences Type II Protein Secretion and Associated Phenotypes in Vibrio cholerae

    doi: 10.3389/fmicb.2019.02780

    Figure Lengend Snippet: Expression of the diNAcBac glycan in V. cholerae results in multiple glycosylated proteins in a PglL Vc -dependent manner. (A) Shown is a representative immunoblot using the anti-diNAcBac antibody (npg1) to detect diNAcBac-glycosylated proteins and the corresponding Kang-stained SDS-gel of whole cell extracts derived from Δ pglL Vc Δ rbmD with or without in trans expression of respective O -OTases PglL Vc or RbmD (+ = pMMBneo-pglL Vc or pMMBneo-rbmD; – = pMMBneo) as well as the diNAcBac glycan (+ = pACYCpglFBCD; – = pACYC184). Only the presence of PglL Vc and diNAcBAc yielded in the detection of multiple bands. (B) Shown is an immunoblot using the npg1-antibody to detect diNAcBac-glycosylated proteins and the corresponding Kang-stained SDS-gel of immunopreciptated samples of Δ pglL Vc Δ rbmD expressing the diNAcBac glycan (pACYCpglFBCD) in the presence (+ = pMMBneo-pglL Vc ) or absence (- = pMMBneo) of PglL Vc . Immunoprecipitation was performed using npg1 immobilized onto Dynabeads coupled with protein G in combination with membrane and periplasmic enriched protein samples from the respective strains. Selected protein bands, which appear to be enriched upon presence of PglL Vc on the SDS-gel and resulted in a decent signal on the immunoblot, were excised and subjected to MS. Identified proteins are indicated on the right with their respective position on the gel, protein identities and accession numbers. (A,B) Lines to the left indicate the molecular masses of the protein standards in kDa.

    Article Snippet: Immunoprecipitation Immunoprecipitation was performed using Dynabeads® Protein G Immunoprecipitation Kit (Invitrogen) according to manufacturer’s manual.

    Techniques: Expressing, Staining, SDS-Gel, Derivative Assay, Immunoprecipitation, Mass Spectrometry

    ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated dynabeads. For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.

    Journal: eLife

    Article Title: LINE-1 protein localization and functional dynamics during the cell cycle

    doi: 10.7554/eLife.30058

    Figure Lengend Snippet: ORF1p antibody comparison. ( A ) Comparison of immunofluorescence stainings using several antibodies against ORF1p and HeLa M2 cells expressing a recoded L1. 4H1 mouse antibody (red) was compared against co-staining of rabbit 4632, rabbit JH74, rabbit JH73g antibodies (all shown in green). DAPI staining (blue) was used to label the nucleus. The lower row of pictures shows a magnification of clustered cells expressing nuclear ORF1p and stained with 4H1 and JH73g antibody. ( B ) Quantification of nuclear and cytoplasmic ORF1p and ORF2p in HeLa cells expressing a recoded L1 (ORFeus) or a native L1 (L1rp) and stained with JH74 or JH73g antibodies in combination with mouse anti-FLAG M2 antibody. The error is expressed as S.E.M. calculated from at least 10 different fields (20X) containing at least 100 cells. ( C ) Western blot of HeLa M2 cells expressing ORFeus, L1rp or no L1 (HeLa) blotted with the indicated antibodies in combination with 4H1 always in green. The same blotting is presented in colors to better show the overlap of red (from rabbit Abs) and green (from mouse Abs) signal, and in gray scale to better show the single bands. Tubulin was used as loading control. 50 μg of protein were loaded in HeLa, ORFeus and L1rp lanes and 25 μg protein in the ORFeus/2 lanes. ( D ) Western blotting of ORF1p and ORF2p immunoprecipitated (IP) form lysates of HeLa cells expressing recoded (ORFeus/LD401) or native (L1rp/MT302) L1. Immunoprecipitation was conducted using IgG control, 4H1 or JH73g conjugated dynabeads. For IPs performed with 4H1 Ab several amounts of immunocomplexes were loaded on the gel: 1/1 = same amount loaded for IgG, 4H1 and JH73g IPs; ½=half of the 1/1 amount; ¼=one fourth of the 1/1 amount; 1/6 = one sixth of the 1/1 amount. Quantification of each band is reported as well as the relative quantification setting the intensity of JH73g IP band as 1.

    Article Snippet: Immunoprecipitations were performed using 5–10 µl of dynabeads conjugated to primary antibodies (Dynabeads Antibody Coupling kit, Life Technologies, prod. number 14311D) incubated with lysates for at least 1 hr nutating at 4°C.

    Techniques: Immunofluorescence, Expressing, Staining, Western Blot, Immunoprecipitation

    Analysis of nuclear fractions. HeLa M2 cells expressing a recoded L1 with a 3XFlagTag on ORF2p C terminus were processed for the isolation of protein cellular fractions using a protein fractionation kit (Thermo prod. number 78840). Upon fractionation ORF2p was immunoprecipitated (IP) from each fraction using FLAG-M2 (SIGMA) conjugated dynabeads. Western blotting analysis for each fraction was performed using the indicated antibodies. (tubulin = cytosol marker, DNA-PK = nuclear soluble marker, calreticulin = membranes marker, H3K4me and H3 = chromatin marker, laminin A/C = nuclear membrane marker, G3BP1 = stress granules marker PCNA-Ub = ubiquitinated PCNA).

    Journal: eLife

    Article Title: LINE-1 protein localization and functional dynamics during the cell cycle

    doi: 10.7554/eLife.30058

    Figure Lengend Snippet: Analysis of nuclear fractions. HeLa M2 cells expressing a recoded L1 with a 3XFlagTag on ORF2p C terminus were processed for the isolation of protein cellular fractions using a protein fractionation kit (Thermo prod. number 78840). Upon fractionation ORF2p was immunoprecipitated (IP) from each fraction using FLAG-M2 (SIGMA) conjugated dynabeads. Western blotting analysis for each fraction was performed using the indicated antibodies. (tubulin = cytosol marker, DNA-PK = nuclear soluble marker, calreticulin = membranes marker, H3K4me and H3 = chromatin marker, laminin A/C = nuclear membrane marker, G3BP1 = stress granules marker PCNA-Ub = ubiquitinated PCNA).

    Article Snippet: Immunoprecipitations were performed using 5–10 µl of dynabeads conjugated to primary antibodies (Dynabeads Antibody Coupling kit, Life Technologies, prod. number 14311D) incubated with lysates for at least 1 hr nutating at 4°C.

    Techniques: Expressing, Isolation, Fractionation, Immunoprecipitation, Western Blot, Marker

    Depletion of NEDD4 compromises assembly of PRC2-Ezh1 complex and H3K27me3 occupancy on mCK, MyoG and MYH8 genomic loci under oxidative stress conditions. a , b Interaction between SUZ12 and EED was determined in scramble and NEDD4 knockdown stable cell lines under normal and oxidative stress conditions. Nuclear extracts from scramble and NEDD4 KD cell lines under normal and stress conditions were immunoprecipitated with SUZ12 antibody, associated protein complexes were eluted with 2XLDS loading buffer. SUZ12 and EED were detected using anti-SUZ12 and anti-EED. Protein A Dynabeads alone were incubated with nuclear extracts and used as mock control. Ponceau S staining was used as loading control. c , d ChIP-qPCR analysis of Ezh1α occupancy and H3K27me3 status on genomic loci of mCK enhancer, MyoG promoter, MYH8 and Neurog1. Chromatin immunoprecipitation (ChIP) was performed using chromatin from scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions against Ezh1α or H3K27me3 antibody. Precipitated DNA were measured using qPCR assay with specific primers corresponding to mCK enhancer, MyoG promoter, MYH8 and NeuroG1 genomic regions. ChIP enrichments are shown as percentage of input. Data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t -test p value. e Transcription level of NEDD4, mCK, MyoG, MYH8 and Atrogin1 were analyzed using RT-qPCR in scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions. Data were expressed as mean ± SD from three biological replicates. GAPDH was normalized to get relative expression of each target. Values above each bar indicate Student’s t -test p value

    Journal: Epigenetics & Chromatin

    Article Title: Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/β-mediated adaptive stress response pathway in skeletal muscle cells

    doi: 10.1186/s13072-019-0322-5

    Figure Lengend Snippet: Depletion of NEDD4 compromises assembly of PRC2-Ezh1 complex and H3K27me3 occupancy on mCK, MyoG and MYH8 genomic loci under oxidative stress conditions. a , b Interaction between SUZ12 and EED was determined in scramble and NEDD4 knockdown stable cell lines under normal and oxidative stress conditions. Nuclear extracts from scramble and NEDD4 KD cell lines under normal and stress conditions were immunoprecipitated with SUZ12 antibody, associated protein complexes were eluted with 2XLDS loading buffer. SUZ12 and EED were detected using anti-SUZ12 and anti-EED. Protein A Dynabeads alone were incubated with nuclear extracts and used as mock control. Ponceau S staining was used as loading control. c , d ChIP-qPCR analysis of Ezh1α occupancy and H3K27me3 status on genomic loci of mCK enhancer, MyoG promoter, MYH8 and Neurog1. Chromatin immunoprecipitation (ChIP) was performed using chromatin from scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions against Ezh1α or H3K27me3 antibody. Precipitated DNA were measured using qPCR assay with specific primers corresponding to mCK enhancer, MyoG promoter, MYH8 and NeuroG1 genomic regions. ChIP enrichments are shown as percentage of input. Data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t -test p value. e Transcription level of NEDD4, mCK, MyoG, MYH8 and Atrogin1 were analyzed using RT-qPCR in scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions. Data were expressed as mean ± SD from three biological replicates. GAPDH was normalized to get relative expression of each target. Values above each bar indicate Student’s t -test p value

    Article Snippet: The immunocomplexes were recovered with magnetic Dynabeads (Protein A; Invitrogen) for 2 h and washed on the wheel at 4 °C for 5 min with Low-Salt (LS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 150 mM NaCl) and High-Salt (HS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 500 mM NaCl).

    Techniques: Stable Transfection, Immunoprecipitation, Incubation, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    CLC-3 is phosphorylated by Ca 2+ /calmodulin kinase II (CaMKII) tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A , tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B , α-CLC-3 was incubated with protein A-conjugated magnetic Dynabeads and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C , CLC-3 is phosphorylated by CaMKII in vitro . Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.

    Journal: The Journal of Physiology

    Article Title: CLC-3 chloride channels moderate long-term potentiation at Schaffer collateral-CA1 synapses

    doi: 10.1113/jphysiol.2012.243485

    Figure Lengend Snippet: CLC-3 is phosphorylated by Ca 2+ /calmodulin kinase II (CaMKII) tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A , tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B , α-CLC-3 was incubated with protein A-conjugated magnetic Dynabeads and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C , CLC-3 is phosphorylated by CaMKII in vitro . Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.

    Article Snippet: Fifty microlitres of protein A-conjugated magnetic Dynabeads (Invitrogen) was incubated with 5 μl α-CLC-3 (Alomone) for 10 min at room temperature with rotation and used to immunoprecipitate CLC-3 from the cell lysate at room temperature for 1 h. Following three washes with PBS, CLC-3 was eluted with Western blot sample buffer (200 m m Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 50 m m EDTA and 100 m m DTT).

    Techniques: Stable Transfection, Transfection, Selection, Plasmid Preparation, Expressing, Western Blot, Incubation, In Vitro, Immunoprecipitation, SDS Page

    Asn-43 mutants still interact with RIP2. Co-immunoprecipitation experiments were performed in HEK293 cells transfected with the constructs detailed in either the presence ( A ) or absence ( B ) of stimulation with 100 ng/ml iE-DAP. Complexes were immunoprecipitated using anti-FLAG antibody immobilized on Protein G-coated Dynabeads and analyzed with the antibodies detailed. Blots are representative of at least three separate experiments. co-purification of E. coli -expressed GST-NOD1 CARD and GB1-RIP2-CARD ( C ) or His-MBP-NOD1-CARD and GB1-RIP2-CARD ( D ). Expressed proteins were pooled during lysis, co-purified with glutathione-Sepharose or amylose resin as appropriate, and detected by Instant Blue (Expedeon) staining. In both C and D , T represents total lysate, and E represents eluted fraction. The position of recombinant proteins is marked. Images are representative of at least three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction *

    doi: 10.1074/jbc.M114.557900

    Figure Lengend Snippet: Asn-43 mutants still interact with RIP2. Co-immunoprecipitation experiments were performed in HEK293 cells transfected with the constructs detailed in either the presence ( A ) or absence ( B ) of stimulation with 100 ng/ml iE-DAP. Complexes were immunoprecipitated using anti-FLAG antibody immobilized on Protein G-coated Dynabeads and analyzed with the antibodies detailed. Blots are representative of at least three separate experiments. co-purification of E. coli -expressed GST-NOD1 CARD and GB1-RIP2-CARD ( C ) or His-MBP-NOD1-CARD and GB1-RIP2-CARD ( D ). Expressed proteins were pooled during lysis, co-purified with glutathione-Sepharose or amylose resin as appropriate, and detected by Instant Blue (Expedeon) staining. In both C and D , T represents total lysate, and E represents eluted fraction. The position of recombinant proteins is marked. Images are representative of at least three independent experiments.

    Article Snippet: Supernatants were incubated with anti-FLAG antibody (Sigma; F3165) immobilized on Protein G-coated magnetic Dynabeads® (Invitrogen) according to the manufacturer's protocol.

    Techniques: Immunoprecipitation, Transfection, Construct, Copurification, Lysis, Purification, Staining, Recombinant

    In vitro interaction between nT and RIII. His-Sumo-tagged nT or RIII was bound to anti-His Dynabeads, and RIII protein pulled down by Dynabeads was analyzed by Western blotting. (Top) Western blotting using an anti-His antibody. Lane 1, His-Sumo tag only

    Journal: Journal of Bacteriology

    Article Title: The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

    doi: 10.1128/JB.00294-16

    Figure Lengend Snippet: In vitro interaction between nT and RIII. His-Sumo-tagged nT or RIII was bound to anti-His Dynabeads, and RIII protein pulled down by Dynabeads was analyzed by Western blotting. (Top) Western blotting using an anti-His antibody. Lane 1, His-Sumo tag only

    Article Snippet: Pulldown assays were conducted according to the instructions in the manufacturer's protocol from the Dynabeads His-Tag Isolation and Pulldown kit (Thermo Fisher Scientific, Rockford, IL).

    Techniques: In Vitro, Western Blot