streptavidin Millipore Search Results


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  • 93
    Millipore streptavidin
    bAMF and anti-AMF-R mAb colocalize on the cell surface. bAMF migrated as a single band in protein blots revealed with <t>HRP-streptavidin</t> (A). Confocal imaging of cell surface labeling of viable NIH-3T3 cells at 4°C with bAMF (B) or anti-AMF-R antibody (C). Confocal images from both fluorescent channels were superimposed (panel D; bAMF in green and AMF-R in red) and revealed a significant degree of colocalization in yellow. Bar, 20 μm.
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin
    bAMF and anti-AMF-R mAb colocalize on the cell surface. bAMF migrated as a single band in protein blots revealed with <t>HRP-streptavidin</t> (A). Confocal imaging of cell surface labeling of viable NIH-3T3 cells at 4°C with bAMF (B) or anti-AMF-R antibody (C). Confocal images from both fluorescent channels were superimposed (panel D; bAMF in green and AMF-R in red) and revealed a significant degree of colocalization in yellow. Bar, 20 μm.
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore alkaline phosphatase conjugated streptavidin
    Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and <t>streptavidin</t> linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore cy3 conjugated streptavidin
    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with <t>Cy3-conjugated</t> <t>streptavidin</t> (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).
    Cy3 Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore cy2 conjugated streptavidin
    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with <t>Cy3-conjugated</t> <t>streptavidin</t> (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).
    Cy2 Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore streptavidin ap
    ESEM images of a microelectrode in each region (A), (B), and (C) after enzyme-NP assembly. Top row: microelectrodes after assembling 31 total alternating layers of 200 nm biotin NPs and <t>streptavidin-AP.</t> Bottom row: microelectrodes after assembling 21 layers of 40 nm biotin NPs and streptavidin-AP.
    Streptavidin Ap, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore streptavidin beads
    Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with <t>streptavidin</t> agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.
    Streptavidin Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 935 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore cy5 labeled streptavidin
    Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with <t>streptavidin</t> agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.
    Cy5 Labeled Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore hrp conjugated streptavidin
    Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with <t>streptavidin-Cy3</t> (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using <t>anti-His-HRP</t> (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.
    Hrp Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore streptavidin pod conjugate
    Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with <t>streptavidin-Cy3</t> (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using <t>anti-His-HRP</t> (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.
    Streptavidin Pod Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore streptavidin atto 655
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin Atto 655, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore atto 594 streptavidin
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Atto 594 Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore streptavidin atto 488
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin Atto 488, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore streptavidin 594
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin 594, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore streptavidin apc
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin Apc, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore streptavidin resin
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin Resin, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore streptavidin tetramer
    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + <t>streptavidin-Atto</t> 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.
    Streptavidin Tetramer, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore atto 633 streptavidin
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Atto 633 Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Millipore streptavidin rpe
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Streptavidin Rpe, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore phycoerythrin streptavidin
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Phycoerythrin Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore streptavidin polyperoxidase
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Streptavidin Polyperoxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore streptavidin sa110
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Streptavidin Sa110, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore lyophilized streptavidin
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Lyophilized Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore streptavidin deac
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Streptavidin Deac, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore streptavidin atto 550
    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto <t>633-Streptavidin</t> (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
    Streptavidin Atto 550, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore tetrameric streptavidin
    Kinetics of inhibition of biotinylated D336C channels by <t>streptavidin.</t> (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.
    Tetrameric Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore tetravalent streptavidin
    ( a ) Library of non- and biotinylated discotics; ( b ) Self-assembling multivalency of the discotics and the <t>streptavidin</t> mutants used in this study.
    Tetravalent Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore streptavidin hrp
    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
    Streptavidin Hrp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore streptavidin strav
    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
    Streptavidin Strav, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti streptavidin antibody
    Phagocytosis Inhibits 3D Migration (A) Marrow macrophages were engineered and mixed with <t>streptavidin</t> beads of 900 nm to 6.7 μm diameter or else with disaggregated tdTom A549 tumors. Beads are opsonized with anti-streptavidin except for a non-targeted control in which SIRPα-inhibited macrophages were incubated with 2.1 μm lacking anti-streptavidin. These were not engulfed. After pre-incubation of the mixtures for up to 150 min, mixtures were plated for 24 hr on top of 5-μm pore transwells. Cells collected from transwell tops and bottoms were stained for CD11b + ,F4/80 + and analyzed by flow cytometry for engulfment (STAR Methods). (B) Bottom/total cell counts gives the %-migrating macrophages, with the non-opsonized control bead result at 0 (n = 3). (C) The %-Eating indicates internalization of at least one bead per macrophages on top of trans-wells (or else just after the pre-incubation) (n = 3). (D) The average number of opsonized beads engulfed per macrophage is ~2 for all bead sizes (n = 3). ). A’PB macrophages were mixed at 10:1 ratio with disaggregated tumor cells or tissue cells. Nearly all A’PB macrophages become tdTom + and rarely migrate, whereas ~50-fold more of the same A’PB macrophages successfully migrate through the pores when incubated with control lung tissue (n = 3). Error bars in (B)–(E) represent mean + SEM.
    Anti Streptavidin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    bAMF and anti-AMF-R mAb colocalize on the cell surface. bAMF migrated as a single band in protein blots revealed with HRP-streptavidin (A). Confocal imaging of cell surface labeling of viable NIH-3T3 cells at 4°C with bAMF (B) or anti-AMF-R antibody (C). Confocal images from both fluorescent channels were superimposed (panel D; bAMF in green and AMF-R in red) and revealed a significant degree of colocalization in yellow. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: bAMF and anti-AMF-R mAb colocalize on the cell surface. bAMF migrated as a single band in protein blots revealed with HRP-streptavidin (A). Confocal imaging of cell surface labeling of viable NIH-3T3 cells at 4°C with bAMF (B) or anti-AMF-R antibody (C). Confocal images from both fluorescent channels were superimposed (panel D; bAMF in green and AMF-R in red) and revealed a significant degree of colocalization in yellow. Bar, 20 μm.

    Article Snippet: Streptavidin conjugated to 10-nm gold particles was purchased from Sigma.

    Techniques: Imaging, Labeling

    Localization of internalized bAMF to AMF-R tubules by confocal microscopy. NIH-3T3 cells were pulse labeled with bAMF at 37°C for 1 h in regular medium (A–F) for 1 h in medium acidified to pH 5.5 to disrupt clathrin-mediated endocytosis (G–I), or in regular medium in the presence of 10-fold excess unlabeled AMF (J–L) before fixation with methanol/acetone. bAMF was revealed with Texas Red-streptavidin (A, D, G, and J) and AMF-R (B, H, and K) or LAMP-1 (E) labeled with the appropriate primary antibodies and FITC-conjugated secondary antibodies. Confocal images from both fluorescent channels were superimposed (panels C, I, and L, bAMF in red and AMF-R in green; panel F, bAMF in red and LAMP-1 in green) and colocalization appears in yellow. Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Localization of internalized bAMF to AMF-R tubules by confocal microscopy. NIH-3T3 cells were pulse labeled with bAMF at 37°C for 1 h in regular medium (A–F) for 1 h in medium acidified to pH 5.5 to disrupt clathrin-mediated endocytosis (G–I), or in regular medium in the presence of 10-fold excess unlabeled AMF (J–L) before fixation with methanol/acetone. bAMF was revealed with Texas Red-streptavidin (A, D, G, and J) and AMF-R (B, H, and K) or LAMP-1 (E) labeled with the appropriate primary antibodies and FITC-conjugated secondary antibodies. Confocal images from both fluorescent channels were superimposed (panels C, I, and L, bAMF in red and AMF-R in green; panel F, bAMF in red and LAMP-1 in green) and colocalization appears in yellow. Bar, 10 μm.

    Article Snippet: Streptavidin conjugated to 10-nm gold particles was purchased from Sigma.

    Techniques: Confocal Microscopy, Labeling

    F). After fixation with methanol/acetone, cells were double labeled with Texas Red-streptavidin to reveal bAMF (A, C, and E) and anti-AMF-R mAb and FITC-conjugated anti-rat secondary antibody to reveal AMF-R labeling (B, D, and F). To ensure that cellular acidification disrupted clathrin-mediated endocytosis of transferrin receptor, NIH-3T3 cells were incubated at 37°C with Texas Red transferrin for 30 min in regular medium (G) or in medium acidified to pH 5.5 (H). Bar, 20 μm.Internalization of bAMF to AMF-R tubules. NIH-3T3 cells were pulse labeled with bAMF at 37°C for 1 h (A and B), for 2 h and chased for 4 h (C and D), or for 1 h in medium acidified to pH 5.5 to disrupt clathrin-mediated endocytosis (E and

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: F). After fixation with methanol/acetone, cells were double labeled with Texas Red-streptavidin to reveal bAMF (A, C, and E) and anti-AMF-R mAb and FITC-conjugated anti-rat secondary antibody to reveal AMF-R labeling (B, D, and F). To ensure that cellular acidification disrupted clathrin-mediated endocytosis of transferrin receptor, NIH-3T3 cells were incubated at 37°C with Texas Red transferrin for 30 min in regular medium (G) or in medium acidified to pH 5.5 (H). Bar, 20 μm.Internalization of bAMF to AMF-R tubules. NIH-3T3 cells were pulse labeled with bAMF at 37°C for 1 h (A and B), for 2 h and chased for 4 h (C and D), or for 1 h in medium acidified to pH 5.5 to disrupt clathrin-mediated endocytosis (E and

    Article Snippet: Streptavidin conjugated to 10-nm gold particles was purchased from Sigma.

    Techniques: Labeling, Incubation

    Electron microscopy of the internalization pathway of bAMF. NIH-3T3 cells were pulsed with bAMF at 37°C for 10 (A, B, and H) or 30 min (C, D, E, F, G, and I). The localization of bAMF was revealed by postembedding labeling with 10-nm gold-conjugated streptavidin. After 10 min, bAMF is localized to cell surface caveolae (A and B). After a 30-min pulse, bAMF is localized to caveolae and smooth vesicles (C and D) and also appears in intracellular membranous tubules (E, F, and G) including distinctive smooth (E) and rough (F) ER elements. bAMF labeling of dense lysosomal structures is also detected (H and I). Bar, 0.1 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Electron microscopy of the internalization pathway of bAMF. NIH-3T3 cells were pulsed with bAMF at 37°C for 10 (A, B, and H) or 30 min (C, D, E, F, G, and I). The localization of bAMF was revealed by postembedding labeling with 10-nm gold-conjugated streptavidin. After 10 min, bAMF is localized to cell surface caveolae (A and B). After a 30-min pulse, bAMF is localized to caveolae and smooth vesicles (C and D) and also appears in intracellular membranous tubules (E, F, and G) including distinctive smooth (E) and rough (F) ER elements. bAMF labeling of dense lysosomal structures is also detected (H and I). Bar, 0.1 μm.

    Article Snippet: Streptavidin conjugated to 10-nm gold particles was purchased from Sigma.

    Techniques: Electron Microscopy, Labeling

    Unfolding induced down-regulation of CD4tl-λ C at the PM. (A) Schematic picture of membrane-tethered and soluble bacteriophage λ model proteins. (B) Immunoblot (IB) analysis of CD4tl and CD4tl-λ/λ C expression before (top) or after CHX chase at 37°C for 1.5 h (bottom). Transiently transfected COS7 cells were cultured at 37°C or 26°C before the CHX chase. (C) Indirect immunostaining of thermally unfolded (37°C) or nativelike (26°C) CD4tl-λ C in nonpermeabilized (top) or permeabilized (bottom) COS7 cells. Calreticulin was used as an ER marker. Bars, 10 µm. (D) The PM density of chimeras was determined by ELISA as described in Materials and methods. (E) Pharmacological characterization of rescued and then unfolded CD4tl-λ C degradation as measured by CHX chase and immunoblotting. Lactacystine (LAC), concanamycin (Con), and bafilomycin A1 (Baf) were added simultaneously with CHX for 3.5 h. NH 4 Cl and chloroquin (Chl) were present during the last 2 h of the CHX chase. Equal amounts of proteins were loaded. (F and G) The PM stability of rescued and then unfolded model proteins at 37°C for 1.5 h was determined at 37°C (F) or in the absence of unfolding at 26°C (G) by ELISA. (H) Internalization of unfolded and rescued model proteins was monitored by Ab uptake at 37°C. Data are expressed as the percentage of initial Ab binding. (I) Recycling efficiency was determined by a biotin-streptavidin sandwich assay as described in Materials and methods and expressed as the percentage of CD4tl. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: Quality control for unfolded proteins at the plasma membrane

    doi: 10.1083/jcb.201006012

    Figure Lengend Snippet: Unfolding induced down-regulation of CD4tl-λ C at the PM. (A) Schematic picture of membrane-tethered and soluble bacteriophage λ model proteins. (B) Immunoblot (IB) analysis of CD4tl and CD4tl-λ/λ C expression before (top) or after CHX chase at 37°C for 1.5 h (bottom). Transiently transfected COS7 cells were cultured at 37°C or 26°C before the CHX chase. (C) Indirect immunostaining of thermally unfolded (37°C) or nativelike (26°C) CD4tl-λ C in nonpermeabilized (top) or permeabilized (bottom) COS7 cells. Calreticulin was used as an ER marker. Bars, 10 µm. (D) The PM density of chimeras was determined by ELISA as described in Materials and methods. (E) Pharmacological characterization of rescued and then unfolded CD4tl-λ C degradation as measured by CHX chase and immunoblotting. Lactacystine (LAC), concanamycin (Con), and bafilomycin A1 (Baf) were added simultaneously with CHX for 3.5 h. NH 4 Cl and chloroquin (Chl) were present during the last 2 h of the CHX chase. Equal amounts of proteins were loaded. (F and G) The PM stability of rescued and then unfolded model proteins at 37°C for 1.5 h was determined at 37°C (F) or in the absence of unfolding at 26°C (G) by ELISA. (H) Internalization of unfolded and rescued model proteins was monitored by Ab uptake at 37°C. Data are expressed as the percentage of initial Ab binding. (I) Recycling efficiency was determined by a biotin-streptavidin sandwich assay as described in Materials and methods and expressed as the percentage of CD4tl. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.

    Article Snippet: CD4 chimeras were internalized for 40 min at 37°C, and the remaining PM biotin-Fab was blocked by 10 µg/ml streptavidin at 0°C (Sigma-Aldrich).

    Techniques: Expressing, Transfection, Cell Culture, Immunostaining, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay

    Branching from curved filaments was observed in vitro. ( A ) Mother filaments (red) immobilized via biotin-streptavidin tethers (asterisks) before nucleation of branches (cyan blue) by Arp2/3 complex (violet). ( B ) Actin branches grow at a branch angle

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

    Article Title: Actin filament curvature biases branching direction

    doi: 10.1073/pnas.1114292109

    Figure Lengend Snippet: Branching from curved filaments was observed in vitro. ( A ) Mother filaments (red) immobilized via biotin-streptavidin tethers (asterisks) before nucleation of branches (cyan blue) by Arp2/3 complex (violet). ( B ) Actin branches grow at a branch angle

    Article Snippet: 300 mTorr oxygen; Plasmod; March Instruments) coverslip and incubated with 10 mg/mL biotinylated BSA (A6043; Sigma-Aldrich) in 80 mM piperazine- N - N′ -bis(2-ethanesulfonic acid) pH 6.9, 1 mM EGTA, and 4 mM MgCl2 for 180 min at 4 °C, and 0.2 mg/mL streptavidin (S4762; Sigma-Aldrich) in 1× calcium and magnesium free PBS (Cellgro; Mediatech) for 30–60 min at 23 °C.

    Techniques: In Vitro

    Discrimination of immobilized DNA homopolymers by αHL pores. ( A ) Schematic representation of a homopolymeric DNA oligonucleotide (blue circles, only the first 25 nucleotides of the 60-nucleotide-long sequence are shown) immobilized inside an αHL pore (gray, cross-section) through the use of a biotin (yellow)–streptavidin (red) linkage. The αHL pore can be divided into 2 halves, each ≈5 nm in length; an upper vestibule located between the cis entrance and the central constriction, and a 14-stranded, transmembrane, antiparallel β-barrel, located between the central constriction and trans exit. The central constriction of 1.4 nm diameter is formed by the Glu-111, Lys-147 (shaded green), and Met-113 side chains contributed by all 7 subunits. ( B and C Left ) Current levels for the WT and E111N/K147N pores when blocked with immobilized poly(dC) and poly(dA) oligonucleotides. ( B and C Right ) Typical event histograms displaying the residual current levels, caused by poly(dC) and poly(dA) oligonucleotide blockages, for the WT and E111N/K147N pores. The mean residual current levels for each oligonucleotide were determined by performing Gaussian fits to the data.

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

    Article Title: Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

    doi: 10.1073/pnas.0901054106

    Figure Lengend Snippet: Discrimination of immobilized DNA homopolymers by αHL pores. ( A ) Schematic representation of a homopolymeric DNA oligonucleotide (blue circles, only the first 25 nucleotides of the 60-nucleotide-long sequence are shown) immobilized inside an αHL pore (gray, cross-section) through the use of a biotin (yellow)–streptavidin (red) linkage. The αHL pore can be divided into 2 halves, each ≈5 nm in length; an upper vestibule located between the cis entrance and the central constriction, and a 14-stranded, transmembrane, antiparallel β-barrel, located between the central constriction and trans exit. The central constriction of 1.4 nm diameter is formed by the Glu-111, Lys-147 (shaded green), and Met-113 side chains contributed by all 7 subunits. ( B and C Left ) Current levels for the WT and E111N/K147N pores when blocked with immobilized poly(dC) and poly(dA) oligonucleotides. ( B and C Right ) Typical event histograms displaying the residual current levels, caused by poly(dC) and poly(dA) oligonucleotide blockages, for the WT and E111N/K147N pores. The mean residual current levels for each oligonucleotide were determined by performing Gaussian fits to the data.

    Article Snippet: Solutions of the biotinylated ssDNAs, at 100 μM in 10 mM Tris·HCl, pH 8.0, 0.1 mM EDTA, were mixed with equal volumes of 25 μM streptavidin (SA) (Sigma-Aldrich) in the same buffer.

    Techniques: Sequencing

    Typical electrophoresis results in 1 mM pH 3.3 sodium citrate (left) and 1 mM pH 9.3 Tris (right). False color fluorescence images (top) show the position of TR-DHPE (red) and streptavidin (green) initially (upper images) and after 10 minutes of applying

    Journal: Analytical chemistry

    Article Title: Supported Bilayer Electrophoresis under Controlled Buffer Conditions

    doi: 10.1021/ac1028819

    Figure Lengend Snippet: Typical electrophoresis results in 1 mM pH 3.3 sodium citrate (left) and 1 mM pH 9.3 Tris (right). False color fluorescence images (top) show the position of TR-DHPE (red) and streptavidin (green) initially (upper images) and after 10 minutes of applying

    Article Snippet: Unlabeled streptavidin was purchased from Sigma.

    Techniques: Electrophoresis, Fluorescence

    The mobility of TR-DHPE and streptavidin in POPC at different ionic strengths. All runs were performed in a pH 7.9, 1:1 sodium citrate/Tris buffer mixture for a total buffer strength of 0.5 mM. NaCl was added to the 0.5 mM buffer to make the 5 or 10 mM

    Journal: Analytical chemistry

    Article Title: Supported Bilayer Electrophoresis under Controlled Buffer Conditions

    doi: 10.1021/ac1028819

    Figure Lengend Snippet: The mobility of TR-DHPE and streptavidin in POPC at different ionic strengths. All runs were performed in a pH 7.9, 1:1 sodium citrate/Tris buffer mixture for a total buffer strength of 0.5 mM. NaCl was added to the 0.5 mM buffer to make the 5 or 10 mM

    Article Snippet: Unlabeled streptavidin was purchased from Sigma.

    Techniques:

    The mobility of streptavidin labeled with an average of 0.3 dyes/molecule as a function of pH at 170 V/cm. All runs were performed at 1 mM buffer concentration in a POPC SLB. Sodium citrate was used under acidic conditions (below pH 6), sodium phosphate

    Journal: Analytical chemistry

    Article Title: Supported Bilayer Electrophoresis under Controlled Buffer Conditions

    doi: 10.1021/ac1028819

    Figure Lengend Snippet: The mobility of streptavidin labeled with an average of 0.3 dyes/molecule as a function of pH at 170 V/cm. All runs were performed at 1 mM buffer concentration in a POPC SLB. Sodium citrate was used under acidic conditions (below pH 6), sodium phosphate

    Article Snippet: Unlabeled streptavidin was purchased from Sigma.

    Techniques: Labeling, Concentration Assay

    The mobility of streptavidin labeled with an average of 4.0 dyes/molecule as a function of pH at 170 V/cm. All runs were performed at 1 mM buffer concentration in a POPC SLB. Sodium citrate was used under acidic conditions (below pH 6), sodium phosphate

    Journal: Analytical chemistry

    Article Title: Supported Bilayer Electrophoresis under Controlled Buffer Conditions

    doi: 10.1021/ac1028819

    Figure Lengend Snippet: The mobility of streptavidin labeled with an average of 4.0 dyes/molecule as a function of pH at 170 V/cm. All runs were performed at 1 mM buffer concentration in a POPC SLB. Sodium citrate was used under acidic conditions (below pH 6), sodium phosphate

    Article Snippet: Unlabeled streptavidin was purchased from Sigma.

    Techniques: Labeling, Concentration Assay

    The mobility of TR-DHPE and streptavidin in POPC (blue) and in a POPC/PEG bilayer (red). The runs were performed in 1 mM sodium citrate (pH 5.2), sodium phosphate (pH 6.5), and Tris (pH 9.3) buffer with an applied potential of 140 V/cm.

    Journal: Analytical chemistry

    Article Title: Supported Bilayer Electrophoresis under Controlled Buffer Conditions

    doi: 10.1021/ac1028819

    Figure Lengend Snippet: The mobility of TR-DHPE and streptavidin in POPC (blue) and in a POPC/PEG bilayer (red). The runs were performed in 1 mM sodium citrate (pH 5.2), sodium phosphate (pH 6.5), and Tris (pH 9.3) buffer with an applied potential of 140 V/cm.

    Article Snippet: Unlabeled streptavidin was purchased from Sigma.

    Techniques:

    Rate analysis of platelet removal from blood. (A) Splenic sequestration of platelets. Crushed spleens from 9/13-week-old WT and RGS18-/- mice (n = 6 per group) were stained for CD42b, and platelets were identified by flow cytometry on the basis of size and CD42b positivity. Percentages of total splenic cell population are shown. (B) Clearance of platelets. Blood cells from 7-week-old mice were biotinylated in vivo by infusion of NHS-biotin, and then blood samples were collected at the time indicated. Biotinylated platelets from WT (black line) and RGS18-/- (red line) mice (n = 9 per group) were identified by flow cytometry on whole blood using PE-Streptavidin and CD41 staining. The stability of the biotinylation was assessed by examining the in vivo biotinylated CD41 - blood cells in WT (green line) and RGS18-/- (yellow line) mice (n = 9 per group). (C) Spontaneous platelet aggregation. Whole blood cells from 9/11-week-old WT and RGS18-/- mice (n = 12 per group) were stained for CD42b, and platelets were identified by flow cytometry on the basis of size and CD42b positivity. Platelet aggregates were monitored by extending the platelet gate so as to include the platelet “smearings” having elevated FSC and FITC fluorescence. Percentages of total cell population are shown. *P

    Journal: PLoS ONE

    Article Title: Regulator of G-Protein Signaling 18 Controls Both Platelet Generation and Function

    doi: 10.1371/journal.pone.0113215

    Figure Lengend Snippet: Rate analysis of platelet removal from blood. (A) Splenic sequestration of platelets. Crushed spleens from 9/13-week-old WT and RGS18-/- mice (n = 6 per group) were stained for CD42b, and platelets were identified by flow cytometry on the basis of size and CD42b positivity. Percentages of total splenic cell population are shown. (B) Clearance of platelets. Blood cells from 7-week-old mice were biotinylated in vivo by infusion of NHS-biotin, and then blood samples were collected at the time indicated. Biotinylated platelets from WT (black line) and RGS18-/- (red line) mice (n = 9 per group) were identified by flow cytometry on whole blood using PE-Streptavidin and CD41 staining. The stability of the biotinylation was assessed by examining the in vivo biotinylated CD41 - blood cells in WT (green line) and RGS18-/- (yellow line) mice (n = 9 per group). (C) Spontaneous platelet aggregation. Whole blood cells from 9/11-week-old WT and RGS18-/- mice (n = 12 per group) were stained for CD42b, and platelets were identified by flow cytometry on the basis of size and CD42b positivity. Platelet aggregates were monitored by extending the platelet gate so as to include the platelet “smearings” having elevated FSC and FITC fluorescence. Percentages of total cell population are shown. *P

    Article Snippet: Samples were stained with FITC rat anti-mouse CD41 (BD Biosciences; 100 ng/µl) and then incubated with PE-Streptavidin (Calbiochem; diluted to 1∶3).

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry, In Vivo, Fluorescence

    (a). Binding of TRAP A domain, A+RII domain and ECD to HepG2 cells. HepG2 cells (approx. 1 × 10 5 ) cells were seeded in 96 well cell culture plates and incubated with PfTRAP fragments in equimolar amounts. Binding was evaluated through ELISA. Results are the mean ± SD of n = 3 experiments. (b). Binding specificity of PfTRAP ECD with hepatoma cell lines. HEK 293 (Kidney cell line), Huh-7 and HepG2 (hepatoma cell lines) cells were incubated with biotinylated PfTRAP ECD. Binding was evaluated through a flow cytometry analysis using Streptavidin-PE. Results are the mean ± SD of n = 3. (c) (i, iii and v) Control FACS data with unstained (black), stained (light grey) and Biotinylated BSA (dark grey) using HepG2 cells, Huh-7 cells and HEK 293 kidney cell lines respectively. (ii, iv and vi) FACS data to evaluate binding of PfTRAP ECD to HepG2 cells, Huh-7 cells and HEK 293 cell lines respectively with the increasing amounts of PfTRAP ECD (grey to thick black) (0.25, 0.5, 1.0 and 2.0 μg) and the stained cells control (thin black).

    Journal: Malaria Journal

    Article Title: Role of Plasmodium falciparum thrombospondin-related anonymous protein in host-cell interactions

    doi: 10.1186/1475-2875-7-63

    Figure Lengend Snippet: (a). Binding of TRAP A domain, A+RII domain and ECD to HepG2 cells. HepG2 cells (approx. 1 × 10 5 ) cells were seeded in 96 well cell culture plates and incubated with PfTRAP fragments in equimolar amounts. Binding was evaluated through ELISA. Results are the mean ± SD of n = 3 experiments. (b). Binding specificity of PfTRAP ECD with hepatoma cell lines. HEK 293 (Kidney cell line), Huh-7 and HepG2 (hepatoma cell lines) cells were incubated with biotinylated PfTRAP ECD. Binding was evaluated through a flow cytometry analysis using Streptavidin-PE. Results are the mean ± SD of n = 3. (c) (i, iii and v) Control FACS data with unstained (black), stained (light grey) and Biotinylated BSA (dark grey) using HepG2 cells, Huh-7 cells and HEK 293 kidney cell lines respectively. (ii, iv and vi) FACS data to evaluate binding of PfTRAP ECD to HepG2 cells, Huh-7 cells and HEK 293 cell lines respectively with the increasing amounts of PfTRAP ECD (grey to thick black) (0.25, 0.5, 1.0 and 2.0 μg) and the stained cells control (thin black).

    Article Snippet: The cells were washed three times with FACS buffer, and streptavidin-PE (Calbiochem) was added to each tube at a dilution of 1: 2000.

    Techniques: Binding Assay, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, FACS, Staining

    Analysis of the RE incubated with streptavidin labeled RNAs (SA-RNA) ( a ) Representative 2D class averages of the RE incubated with different SA-RNA substrates (SA-RNA08, SA-RNA16, SA-RNA24, SA-RNA36, SA-RNA47 and SA-RNA50). Additional SA densities compared to Apo-RE class-averages are marked with red circles. The scale bar represents 20 nm. ( b ) Distribution of particles belonging to the class-average views with or without SA among particles for each sample set. ( c-f ) Front ( c-d ), back( e ) and bottom ( f ) views of the SA-RNA47-RE 3D model, where d and e both have a tetrameric streptavidin crystal structure (PDB 1NDJ) 37 docked into the electron densities. ( g-h ) 3D models of the RE-long ( g ) and RE-short ( h ) conformations in bottom view, respectively.

    Journal: Nature structural & molecular biology

    Article Title: Visualization of Distinct Substrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy

    doi: 10.1038/nsmb.2736

    Figure Lengend Snippet: Analysis of the RE incubated with streptavidin labeled RNAs (SA-RNA) ( a ) Representative 2D class averages of the RE incubated with different SA-RNA substrates (SA-RNA08, SA-RNA16, SA-RNA24, SA-RNA36, SA-RNA47 and SA-RNA50). Additional SA densities compared to Apo-RE class-averages are marked with red circles. The scale bar represents 20 nm. ( b ) Distribution of particles belonging to the class-average views with or without SA among particles for each sample set. ( c-f ) Front ( c-d ), back( e ) and bottom ( f ) views of the SA-RNA47-RE 3D model, where d and e both have a tetrameric streptavidin crystal structure (PDB 1NDJ) 37 docked into the electron densities. ( g-h ) 3D models of the RE-long ( g ) and RE-short ( h ) conformations in bottom view, respectively.

    Article Snippet: Gel shifting and pull down assays Streptavidin (SA) was purchased from Sigma and HRP-coupled streptavidin (HRP-SA) was from Abicom.

    Techniques: Incubation, Labeling

    hChlR1 catalyzes streptavidin displacement from biotinylated oligonucleotide. A , the indicated concentrations of hChlR1 were incubated with 1 m m ATP and 0.5 n m DNA substrate that had streptavidin bound to the covalently linked biotin moiety residing 52

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical Characterization of Warsaw Breakage Syndrome Helicase

    doi: 10.1074/jbc.M111.276022

    Figure Lengend Snippet: hChlR1 catalyzes streptavidin displacement from biotinylated oligonucleotide. A , the indicated concentrations of hChlR1 were incubated with 1 m m ATP and 0.5 n m DNA substrate that had streptavidin bound to the covalently linked biotin moiety residing 52

    Article Snippet: For streptavidin displacement reactions, 10 fmol of DNA substrate was preincubated with 100 n m streptavidin (Sigma) for 10 min at 37 °C.

    Techniques: Incubation

    Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and streptavidin linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.

    Journal: PLoS ONE

    Article Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain

    doi: 10.1371/journal.pone.0000515

    Figure Lengend Snippet: Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and streptavidin linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.

    Article Snippet: Biotinylated NMB-1 was visualized by incubating cultures in alkaline phosphatase-conjugated streptavidin (Chemicon); alkaline phosphatase colour reaction was carried out using BCIP/NBT kit from Vector Laboratories according to the manufacturer's instructions.

    Techniques: Staining, Binding Assay

    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).

    Journal: BMC Cancer

    Article Title: Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

    doi: 10.1186/1471-2407-13-476

    Figure Lengend Snippet: Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).

    Article Snippet: Negative controls included only Cy3-conjugated streptavidin and FITC-conjugated AffiniPure donkey anti-mouse and also Streptomyces hyaluronidase (30 min RT, 10 U/ml, Sigma-Aldrich) treatment for the HA staining.

    Techniques: Expressing, Quantitative RT-PCR, Incubation

    ESEM images of a microelectrode in each region (A), (B), and (C) after enzyme-NP assembly. Top row: microelectrodes after assembling 31 total alternating layers of 200 nm biotin NPs and streptavidin-AP. Bottom row: microelectrodes after assembling 21 layers of 40 nm biotin NPs and streptavidin-AP.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Electric-Field-Directed Self-Assembly of Active Enzyme-Nanoparticle Structures

    doi: 10.1155/2012/178487

    Figure Lengend Snippet: ESEM images of a microelectrode in each region (A), (B), and (C) after enzyme-NP assembly. Top row: microelectrodes after assembling 31 total alternating layers of 200 nm biotin NPs and streptavidin-AP. Bottom row: microelectrodes after assembling 21 layers of 40 nm biotin NPs and streptavidin-AP.

    Article Snippet: For chips with layer structures assembled using streptavidin-AP, the chips were washed with 100 mM L-histidine buffer and then 15 μ L of CDP-star chemiluminescent reagent (Sigma) was dispensed onto the chips.

    Techniques:

    Plot showing the calculated mean integrated density per microelectrode for microelectrodes in regions B and C in bienzyme layer structures of 9, 19, 29, 39, and 47 total layers of 200 nm biotin NPs with alternate enzyme layer addressing of GOx-avidin and streptavidin-HRP.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Electric-Field-Directed Self-Assembly of Active Enzyme-Nanoparticle Structures

    doi: 10.1155/2012/178487

    Figure Lengend Snippet: Plot showing the calculated mean integrated density per microelectrode for microelectrodes in regions B and C in bienzyme layer structures of 9, 19, 29, 39, and 47 total layers of 200 nm biotin NPs with alternate enzyme layer addressing of GOx-avidin and streptavidin-HRP.

    Article Snippet: For chips with layer structures assembled using streptavidin-AP, the chips were washed with 100 mM L-histidine buffer and then 15 μ L of CDP-star chemiluminescent reagent (Sigma) was dispensed onto the chips.

    Techniques: Avidin-Biotin Assay

    Plot of the relative intensity of chemiluminescent signal obtained from chips addressed with 0, 11, 21, and 31 layers of 200 nm biotin NPs and GOx-avidin or 0, 11, and 31 layers with streptavidin-AP.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Electric-Field-Directed Self-Assembly of Active Enzyme-Nanoparticle Structures

    doi: 10.1155/2012/178487

    Figure Lengend Snippet: Plot of the relative intensity of chemiluminescent signal obtained from chips addressed with 0, 11, 21, and 31 layers of 200 nm biotin NPs and GOx-avidin or 0, 11, and 31 layers with streptavidin-AP.

    Article Snippet: For chips with layer structures assembled using streptavidin-AP, the chips were washed with 100 mM L-histidine buffer and then 15 μ L of CDP-star chemiluminescent reagent (Sigma) was dispensed onto the chips.

    Techniques: Avidin-Biotin Assay

    Coupling of bi-enzyme NP layers. The incorporation of both streptavidin-HRP and GOx-avidin into the same layer structure may allow for chemical coupling of the layers. The oxidation of glucose by GOx produces hydrogen peroxide which is then a substrate for the chemiluminescent oxidation of luminol, which generates light that can be detected.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Electric-Field-Directed Self-Assembly of Active Enzyme-Nanoparticle Structures

    doi: 10.1155/2012/178487

    Figure Lengend Snippet: Coupling of bi-enzyme NP layers. The incorporation of both streptavidin-HRP and GOx-avidin into the same layer structure may allow for chemical coupling of the layers. The oxidation of glucose by GOx produces hydrogen peroxide which is then a substrate for the chemiluminescent oxidation of luminol, which generates light that can be detected.

    Article Snippet: For chips with layer structures assembled using streptavidin-AP, the chips were washed with 100 mM L-histidine buffer and then 15 μ L of CDP-star chemiluminescent reagent (Sigma) was dispensed onto the chips.

    Techniques: Avidin-Biotin Assay

    Electric-field-directed assembly (layering) of biomolecule NPs by different binding mechanisms: (a) NP layering with alternate biotin (blue)-functionalized NPs and streptavidin (yellow)-functionalized NPs. (b) NP layering by hybridization of complementary DNA sequences. (c) NP layering of biotin-functionalized NPs with streptavidin-functionalized enzymes (brown) (image not to scale).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Electric-Field-Directed Self-Assembly of Active Enzyme-Nanoparticle Structures

    doi: 10.1155/2012/178487

    Figure Lengend Snippet: Electric-field-directed assembly (layering) of biomolecule NPs by different binding mechanisms: (a) NP layering with alternate biotin (blue)-functionalized NPs and streptavidin (yellow)-functionalized NPs. (b) NP layering by hybridization of complementary DNA sequences. (c) NP layering of biotin-functionalized NPs with streptavidin-functionalized enzymes (brown) (image not to scale).

    Article Snippet: For chips with layer structures assembled using streptavidin-AP, the chips were washed with 100 mM L-histidine buffer and then 15 μ L of CDP-star chemiluminescent reagent (Sigma) was dispensed onto the chips.

    Techniques: Binding Assay, Hybridization

    Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with streptavidin agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.

    Journal: Nucleic Acids Research

    Article Title: Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly

    doi: 10.1093/nar/gkx545

    Figure Lengend Snippet: Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with streptavidin agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.

    Article Snippet: Histone peptide binding assay Biotinylated histone peptides (Epicypher) were diluted to 1μM in 0.1% (v/v) triton-X100 in PBS and incubated with streptavidin beads (Novagen) for 3 h at room temperature.

    Techniques: Incubation, Recombinant, SDS Page, Peptide Microarray, Methylation, Modification

    3′Biot-mH2a/5m pre-mRNA is resistant to cleavage but assembles into processing complexes. ( A ) A schematic representation of chemically synthesized mouse-specific 3′Biot-mH2a/5m pre-mRNA (64-nt). The major cleavage site (located 5 nt downstream of the stem) and 2 nt on each side of the major cleavage site are modified with a 2′O-methyl group. Biotin is placed at the 3′ end. ( B ) In vitro processing of 3′Biot-mH2a/5m (bottom) and mH2a-614 (top) pre-mRNAs. mH2a-614 (85 nt) was generated by T7 transcription and contains the same HDE as 3′Biot-mH2a/5m but lacks biotin and modified nucleotides. Each pre-mRNA was labeled at the 5′ end with 32 P and incubated in a mouse nuclear extract for 5, 15 and 30 min, as indicated. Probe alone is shown in lane 1. Numbers to the right indicate the length of the input pre-mRNA and the upstream cleavage product. ( C ) 3′Biot-mH2a/5m was incubated with a mouse myeloma nuclear extract (Mm NE) containing recombinant N-terminal FLASH (FLASH/N, amino acids 53–138) fused to GST. Assembled processing complexes were purified on streptavidin beads and analyzed by western blotting using specific antibodies (lane 1). In lane 2, formation of the processing complexes was blocked by excess SL RNA and αU7 oligonucleotide complementary to the 5′ end of mouse U7 snRNA.

    Journal: Nucleic Acids Research

    Article Title: Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

    doi: 10.1093/nar/gky133

    Figure Lengend Snippet: 3′Biot-mH2a/5m pre-mRNA is resistant to cleavage but assembles into processing complexes. ( A ) A schematic representation of chemically synthesized mouse-specific 3′Biot-mH2a/5m pre-mRNA (64-nt). The major cleavage site (located 5 nt downstream of the stem) and 2 nt on each side of the major cleavage site are modified with a 2′O-methyl group. Biotin is placed at the 3′ end. ( B ) In vitro processing of 3′Biot-mH2a/5m (bottom) and mH2a-614 (top) pre-mRNAs. mH2a-614 (85 nt) was generated by T7 transcription and contains the same HDE as 3′Biot-mH2a/5m but lacks biotin and modified nucleotides. Each pre-mRNA was labeled at the 5′ end with 32 P and incubated in a mouse nuclear extract for 5, 15 and 30 min, as indicated. Probe alone is shown in lane 1. Numbers to the right indicate the length of the input pre-mRNA and the upstream cleavage product. ( C ) 3′Biot-mH2a/5m was incubated with a mouse myeloma nuclear extract (Mm NE) containing recombinant N-terminal FLASH (FLASH/N, amino acids 53–138) fused to GST. Assembled processing complexes were purified on streptavidin beads and analyzed by western blotting using specific antibodies (lane 1). In lane 2, formation of the processing complexes was blocked by excess SL RNA and αU7 oligonucleotide complementary to the 5′ end of mouse U7 snRNA.

    Article Snippet: The assembly of processing complexes and their immobilization on streptavidin beads (Sigma) were carried out as described previously , with minor modifications.

    Techniques: Synthesized, Modification, In Vitro, Generated, Labeling, Incubation, Recombinant, Purification, Western Blot

    Drosophila nuclear proteins that bind pcB-dH3/5m pre-mRNA. ( A ) A schematic representation of chemically synthesized Drosophila -specific pcB-dH3/5m pre-mRNA (63 nt). ( B ) pcB-dH3/5m pre-mRNA was incubated with two different batches of Drosophila Kc nuclear extract in the absence or in the presence of SL RNA and αU7 oligonucleotide complementary to the 5′ end of Drosophila U7 snRNA. Proteins bound to pcB-dH3/5m pre-mRNA were immobilized on streptavidin beads and eluted by irradiation with long wave UV. A fraction of each UV-eluted supernatant was analyzed by silver staining (top panel) or western blotting (bottom panels). ( C ) The same fraction was also analyzed by mass spectrometry. Proteins with the 10 highest Mascot scores for Kc NE1 are shown above the dashed line, with arrowheads indicating proteins that interact with histone pre-mRNA in the presence of SL RNA and αU7 oligonucleotide. The remaining components of the U7 snRNP and their overall ranking among all identified proteins (numbering for Kc NE1) are shown below the dashed line. Note that carboxylase results from contamination of the UV eluate with a small amount of streptavidin beads.

    Journal: Nucleic Acids Research

    Article Title: Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

    doi: 10.1093/nar/gky133

    Figure Lengend Snippet: Drosophila nuclear proteins that bind pcB-dH3/5m pre-mRNA. ( A ) A schematic representation of chemically synthesized Drosophila -specific pcB-dH3/5m pre-mRNA (63 nt). ( B ) pcB-dH3/5m pre-mRNA was incubated with two different batches of Drosophila Kc nuclear extract in the absence or in the presence of SL RNA and αU7 oligonucleotide complementary to the 5′ end of Drosophila U7 snRNA. Proteins bound to pcB-dH3/5m pre-mRNA were immobilized on streptavidin beads and eluted by irradiation with long wave UV. A fraction of each UV-eluted supernatant was analyzed by silver staining (top panel) or western blotting (bottom panels). ( C ) The same fraction was also analyzed by mass spectrometry. Proteins with the 10 highest Mascot scores for Kc NE1 are shown above the dashed line, with arrowheads indicating proteins that interact with histone pre-mRNA in the presence of SL RNA and αU7 oligonucleotide. The remaining components of the U7 snRNP and their overall ranking among all identified proteins (numbering for Kc NE1) are shown below the dashed line. Note that carboxylase results from contamination of the UV eluate with a small amount of streptavidin beads.

    Article Snippet: The assembly of processing complexes and their immobilization on streptavidin beads (Sigma) were carried out as described previously , with minor modifications.

    Techniques: Synthesized, Incubation, Irradiation, Silver Staining, Western Blot, Mass Spectrometry

    Effects of increasing the base pairing potential between histone pre-mRNA and Drosophila U7 snRNA. ( A ) A proposed base pair alignment between the HDE of dH3 Ext (left) and dH3/21bp (right) pre-mRNAs and Drosophila U7 snRNA. The HDE starts 10 nt 3′ of the cleavage site and contains the GAGA sequence (underlined) that is essential for processing and is predicted to form an obligatory duplex with the UCUC motif (indicated with a double-headed arrow) conserved in most U7 snRNAs. ( B ) Processing of 5′-labeled dH3 Ext (top) and dH3/21bp (bottom) pre-mRNAs in a Kc nuclear extract either lacking (lane 2) or containing indicated competitors (lanes 3 and 4). The input RNA is shown in lane 1. Length of the uncut pre-mRNAs and the upstream cleavage products (cut) in nucleotides is shown to the right ( C ). dH3 Ext and dH3/21bp pre-mRNAs were annealed to pcB/21mer and the resultant duplexes incubated with a Kc nuclear extract in the absence or presence of oligonucleotide competitors, as indicated. The assembled processing complexes were immobilized on streptavidin beads and analyzed by western blotting for the presence of SLBP and major subunits of the U7 snRNP. ( D ) A hypothetical model explaining essential role of Drosophila in processing. The three α helices of the RBD of Drosophila SLBP are depicted as rectangles and the repeated SD motif is shown at the C-terminal region. In the absence of Drosophila SLBP, U7 snRNP inefficiently binds histone pre-mRNA (blue line) via limited base paring between U7 snRNA (gray line) and the HDE. The bound U7 snRNP in spite of containing all necessary subunits is unable to carry out the cleavage reaction, possibly due to the excessive flexibility of the pre-mRNA substrate (illustrated by multiple arrows). SLBP bound to the stem-loop uses helix B and the C-terminal region to promote the recruitment of the U7 snRNP to the HDE, likely by contacting Lsm11 bound to FLASH and possibly Lsm10. The same network of interactions may result in a major structural rearrangement of the processing complex and stable alignment of the active site of CPSF73 (indicated with an arrow) with the pre-mRNA.

    Journal: Nucleic Acids Research

    Article Title: Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

    doi: 10.1093/nar/gky133

    Figure Lengend Snippet: Effects of increasing the base pairing potential between histone pre-mRNA and Drosophila U7 snRNA. ( A ) A proposed base pair alignment between the HDE of dH3 Ext (left) and dH3/21bp (right) pre-mRNAs and Drosophila U7 snRNA. The HDE starts 10 nt 3′ of the cleavage site and contains the GAGA sequence (underlined) that is essential for processing and is predicted to form an obligatory duplex with the UCUC motif (indicated with a double-headed arrow) conserved in most U7 snRNAs. ( B ) Processing of 5′-labeled dH3 Ext (top) and dH3/21bp (bottom) pre-mRNAs in a Kc nuclear extract either lacking (lane 2) or containing indicated competitors (lanes 3 and 4). The input RNA is shown in lane 1. Length of the uncut pre-mRNAs and the upstream cleavage products (cut) in nucleotides is shown to the right ( C ). dH3 Ext and dH3/21bp pre-mRNAs were annealed to pcB/21mer and the resultant duplexes incubated with a Kc nuclear extract in the absence or presence of oligonucleotide competitors, as indicated. The assembled processing complexes were immobilized on streptavidin beads and analyzed by western blotting for the presence of SLBP and major subunits of the U7 snRNP. ( D ) A hypothetical model explaining essential role of Drosophila in processing. The three α helices of the RBD of Drosophila SLBP are depicted as rectangles and the repeated SD motif is shown at the C-terminal region. In the absence of Drosophila SLBP, U7 snRNP inefficiently binds histone pre-mRNA (blue line) via limited base paring between U7 snRNA (gray line) and the HDE. The bound U7 snRNP in spite of containing all necessary subunits is unable to carry out the cleavage reaction, possibly due to the excessive flexibility of the pre-mRNA substrate (illustrated by multiple arrows). SLBP bound to the stem-loop uses helix B and the C-terminal region to promote the recruitment of the U7 snRNP to the HDE, likely by contacting Lsm11 bound to FLASH and possibly Lsm10. The same network of interactions may result in a major structural rearrangement of the processing complex and stable alignment of the active site of CPSF73 (indicated with an arrow) with the pre-mRNA.

    Article Snippet: The assembly of processing complexes and their immobilization on streptavidin beads (Sigma) were carried out as described previously , with minor modifications.

    Techniques: Sequencing, Labeling, Incubation, Western Blot

    Cleavage activity of Drosophila processing complexes assembled in the absence of SLBP. ( A ) dH3 Ext duplex was incubated in a Kc nuclear extract in the absence or in the presence of indicated competitors and the UV-eluted samples were visualized by silver staining (left) and analyzed by mass spectrometry following a brief electrophoresis into 4–12 gel SDS/polyacrylamide gel (right). Only SLBP and components of the U7 snRNP are listed. They all fail to interact with dH3 pre-mRNA in the presence of SL RNA or αU7 oligonucleotide. A small amount of contaminating streptavidin (SA) in lanes 1 and 3 is indicated with an arrow. ( B and C ) SLBP-free Drosophila processing complexes assembled on dH3 Ext duplex (panel B) or 3′Biot-dH3/2m (panel C) in a nuclear extract containing the SL RNA (as in lane 3 of panel A) were immobilized on streptavidin beads, washed and tested for the ability to support cleavage at 22°C (lanes 2–5 in both panels) either alone or in the presence of Kc nuclear extract or indicated SLBP variants. The aliquots incubated on ice are shown in lane 1 in both panels.

    Journal: Nucleic Acids Research

    Article Title: Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

    doi: 10.1093/nar/gky133

    Figure Lengend Snippet: Cleavage activity of Drosophila processing complexes assembled in the absence of SLBP. ( A ) dH3 Ext duplex was incubated in a Kc nuclear extract in the absence or in the presence of indicated competitors and the UV-eluted samples were visualized by silver staining (left) and analyzed by mass spectrometry following a brief electrophoresis into 4–12 gel SDS/polyacrylamide gel (right). Only SLBP and components of the U7 snRNP are listed. They all fail to interact with dH3 pre-mRNA in the presence of SL RNA or αU7 oligonucleotide. A small amount of contaminating streptavidin (SA) in lanes 1 and 3 is indicated with an arrow. ( B and C ) SLBP-free Drosophila processing complexes assembled on dH3 Ext duplex (panel B) or 3′Biot-dH3/2m (panel C) in a nuclear extract containing the SL RNA (as in lane 3 of panel A) were immobilized on streptavidin beads, washed and tested for the ability to support cleavage at 22°C (lanes 2–5 in both panels) either alone or in the presence of Kc nuclear extract or indicated SLBP variants. The aliquots incubated on ice are shown in lane 1 in both panels.

    Article Snippet: The assembly of processing complexes and their immobilization on streptavidin beads (Sigma) were carried out as described previously , with minor modifications.

    Techniques: Activity Assay, Incubation, Silver Staining, Mass Spectrometry, Electrophoresis

    Cleavage activity of immobilized mouse and Drosophila processing complexes. ( A ) A schematic representation of chemically synthesized mouse-specific 3′Biot-mH2a/2m pre-mRNA (64 nt). The two 2′O-methyl groups placed immediately upstream of the HDE have no effect on endonucleolytic cleavage but prevent subsequent 5′-3′ degradation of the downstream cleavage product. Biotin is placed at the 3′ end and the HDE is modified by two point mutations to maximize its base pair interaction with U7 snRNA (HDE max). ( B ) In vitro processing of 3′Biot-mH2a/2m pre-mRNA in a mouse nuclear extract alone (lane 1) or containing indicated reagents: αU7 oligonucleotide (10 ng/μl), SL RNA (10 ng/μl) or human baculovirus-expressed SLBP (25 ng/μl). ( C ) Mouse processing complexes (lanes 1–4) were assembled on ice by incubating 32 P-labeled 3′Biot-mH2a/2m pre-mRNA with a mouse nuclear extract containing recombinant N-terminal FLASH. During this step, significant cleavage occurred reducing the amount of fully assembled processing complexes (lane 1). Length in nucleotides of the input 3′Biot-mH2a/2m pre-mRNA and of the upstream cleavage product in nucleotides is shown to the right. The assembled complexes were immobilized on streptavidin beads, thoroughly washed and analyzed by western blotting for the presence of SLBP and selected subunits of the U7 snRNP (lane 2) or incubated with a gentle agitation at indicated temperatures to determine their ability to support cleavage (lanes 3 and 4). As explained above, in mouse nuclear extracts SLBP is partially degraded and in SDS gels migrates as a group of closely spaced bands rather than a single full length species of ∼45 kDa. ( D ) Drosophila processing complexes were assembled on Drosophila -specific 3′Biot-dH3/2m pre-mRNA. The complexes were immobilized on streptavidin beads, washed and incubated with a gentle agitation at 0°C (lane 1) or 22°C (lane 2) to determine their ability to support cleavage.

    Journal: Nucleic Acids Research

    Article Title: Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

    doi: 10.1093/nar/gky133

    Figure Lengend Snippet: Cleavage activity of immobilized mouse and Drosophila processing complexes. ( A ) A schematic representation of chemically synthesized mouse-specific 3′Biot-mH2a/2m pre-mRNA (64 nt). The two 2′O-methyl groups placed immediately upstream of the HDE have no effect on endonucleolytic cleavage but prevent subsequent 5′-3′ degradation of the downstream cleavage product. Biotin is placed at the 3′ end and the HDE is modified by two point mutations to maximize its base pair interaction with U7 snRNA (HDE max). ( B ) In vitro processing of 3′Biot-mH2a/2m pre-mRNA in a mouse nuclear extract alone (lane 1) or containing indicated reagents: αU7 oligonucleotide (10 ng/μl), SL RNA (10 ng/μl) or human baculovirus-expressed SLBP (25 ng/μl). ( C ) Mouse processing complexes (lanes 1–4) were assembled on ice by incubating 32 P-labeled 3′Biot-mH2a/2m pre-mRNA with a mouse nuclear extract containing recombinant N-terminal FLASH. During this step, significant cleavage occurred reducing the amount of fully assembled processing complexes (lane 1). Length in nucleotides of the input 3′Biot-mH2a/2m pre-mRNA and of the upstream cleavage product in nucleotides is shown to the right. The assembled complexes were immobilized on streptavidin beads, thoroughly washed and analyzed by western blotting for the presence of SLBP and selected subunits of the U7 snRNP (lane 2) or incubated with a gentle agitation at indicated temperatures to determine their ability to support cleavage (lanes 3 and 4). As explained above, in mouse nuclear extracts SLBP is partially degraded and in SDS gels migrates as a group of closely spaced bands rather than a single full length species of ∼45 kDa. ( D ) Drosophila processing complexes were assembled on Drosophila -specific 3′Biot-dH3/2m pre-mRNA. The complexes were immobilized on streptavidin beads, washed and incubated with a gentle agitation at 0°C (lane 1) or 22°C (lane 2) to determine their ability to support cleavage.

    Article Snippet: The assembly of processing complexes and their immobilization on streptavidin beads (Sigma) were carried out as described previously , with minor modifications.

    Techniques: Activity Assay, Synthesized, Modification, In Vitro, Labeling, Recombinant, Western Blot, Incubation

    FOXP1 is SUMOylated at K670. ( A ) DIV7 cortical neuronal cultures were lysed in buffer with or without 2% SDS and immunoblotted with FOXP1 antibody. Under denaturing conditions a prominent additional higher molecular weight FOXP1 band was observed. ( B ) Following lysis in 2% SDS containing buffer, the SDS was diluted to 0.1% and FOXP1 immunoprecipitated and incubated with GST, GST-WT-Senp1 catalytic domain or GST-Senp1-C603S catalytic domain. Immunoblots with FOXP1 antibody (top panel) shows the higher Mr band is removed by Senp1 treatment consistent with it being SUMOylated FOXP1. A high exposure (top left panel) of the input shows the presence of the modified-FOXP1. Lower panel shows equal levels of purified GST-SENP proteins by coomassie staining. ( C ) Immunoprecipitation was performed as in B, using a SUMO-1 antibody, followed by immunoblotting with either FOXP1 (upper panel) or SUMO1 (lower panel) antibodies. SUMO1-modified FOXP1 corresponds to the high Mr band in the input. Note the enrichment of SUMO1-ylated proteins (seen as a smear) in the IP lane but not the control IgG. ( D ) Schematic of FOXP1 showing the putative SUMOylated lysine. TRD, transcriptional repression domain; LZ, leucine zipper; FOX, FOX homolgy domain; NLS, nuclear localisation signal. ( E ) FOXP1 is SUMOylated at lysine 670. HEK293T cells were transfected with either TAP-FOXP1-WT or the mutant TAP-FOXP1-K670R, lysed in buffer containing NEM, to inhibit SUMO proteases, and precipitated using strepavidin beads before subsequent immunoblotting with anti-SUMO1 (top panel) or anti-SBP (lower panel) antibodies.

    Journal: Scientific Reports

    Article Title: SUMOylation of FOXP1 regulates transcriptional repression via CtBP1 to drive dendritic morphogenesis

    doi: 10.1038/s41598-017-00707-6

    Figure Lengend Snippet: FOXP1 is SUMOylated at K670. ( A ) DIV7 cortical neuronal cultures were lysed in buffer with or without 2% SDS and immunoblotted with FOXP1 antibody. Under denaturing conditions a prominent additional higher molecular weight FOXP1 band was observed. ( B ) Following lysis in 2% SDS containing buffer, the SDS was diluted to 0.1% and FOXP1 immunoprecipitated and incubated with GST, GST-WT-Senp1 catalytic domain or GST-Senp1-C603S catalytic domain. Immunoblots with FOXP1 antibody (top panel) shows the higher Mr band is removed by Senp1 treatment consistent with it being SUMOylated FOXP1. A high exposure (top left panel) of the input shows the presence of the modified-FOXP1. Lower panel shows equal levels of purified GST-SENP proteins by coomassie staining. ( C ) Immunoprecipitation was performed as in B, using a SUMO-1 antibody, followed by immunoblotting with either FOXP1 (upper panel) or SUMO1 (lower panel) antibodies. SUMO1-modified FOXP1 corresponds to the high Mr band in the input. Note the enrichment of SUMO1-ylated proteins (seen as a smear) in the IP lane but not the control IgG. ( D ) Schematic of FOXP1 showing the putative SUMOylated lysine. TRD, transcriptional repression domain; LZ, leucine zipper; FOX, FOX homolgy domain; NLS, nuclear localisation signal. ( E ) FOXP1 is SUMOylated at lysine 670. HEK293T cells were transfected with either TAP-FOXP1-WT or the mutant TAP-FOXP1-K670R, lysed in buffer containing NEM, to inhibit SUMO proteases, and precipitated using strepavidin beads before subsequent immunoblotting with anti-SUMO1 (top panel) or anti-SBP (lower panel) antibodies.

    Article Snippet: Mass spectrometry HEK293T cells were transfected with either empty TAP vector or TAP-FOXP1 and lysed 48 hr after transfection in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCL, 0.5% NP-40 and Roche protease inhibitor cocktail) before pull-down of the TAP tag using strepavidin beads (Sigma) for 1 hr.

    Techniques: Molecular Weight, Lysis, Immunoprecipitation, Incubation, Western Blot, Modification, Purification, Staining, Transfection, Mutagenesis

    Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with streptavidin-Cy3 (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using anti-His-HRP (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains

    doi: 10.1093/nar/gky818

    Figure Lengend Snippet: Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with streptavidin-Cy3 (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using anti-His-HRP (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.

    Article Snippet: Expression of the BAP-tagged proteins was detected with an HRP-conjugated anti-polyHistidine antibody at 1:4000 (Sigma A7058) and biotinylated proteins were visualized with an HRP-conjugated streptavidin at 1:1000 (Sigma S2438).

    Techniques: Transfection, Staining, Labeling, Western Blot

    CD5 and CD6 ectodomains bind PSC tegumental antigens. ( A ) Biotin-labeled rshCD5, rshCD6 and BSA protein solutions (20 μg/mL) were sequentially incubated (4 times) with PSC suspensions (5,000 PSC; viability ≥90%), and pellets and solutions run on SDS-PAGE and further Western blotted with HRP-streptavidin. Lanes 1 and 2: protein solutions before (0x) and after 4 sequential incubations with PSC (4x), respectively. Lanes 3 to 7: PSC pellets after 0x to 4x sequential incubations. ( B ) ELISA assays showing the binding of increasing amounts of biotinylated rshCD5, rshCD6 and BSA proteins to PSEx-coated plates. ( C ) ELISA assays showing the binding of supernatants from sequential PSC incubations of biotin-labeled rshCD5, rshCD6 and BSA depicted in ( A ), to PSEx-coated plates. N.D. not detected. (*) Significant differences (Student’s t-test, P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis

    doi: 10.1371/journal.pntd.0006891

    Figure Lengend Snippet: CD5 and CD6 ectodomains bind PSC tegumental antigens. ( A ) Biotin-labeled rshCD5, rshCD6 and BSA protein solutions (20 μg/mL) were sequentially incubated (4 times) with PSC suspensions (5,000 PSC; viability ≥90%), and pellets and solutions run on SDS-PAGE and further Western blotted with HRP-streptavidin. Lanes 1 and 2: protein solutions before (0x) and after 4 sequential incubations with PSC (4x), respectively. Lanes 3 to 7: PSC pellets after 0x to 4x sequential incubations. ( B ) ELISA assays showing the binding of increasing amounts of biotinylated rshCD5, rshCD6 and BSA proteins to PSEx-coated plates. ( C ) ELISA assays showing the binding of supernatants from sequential PSC incubations of biotin-labeled rshCD5, rshCD6 and BSA depicted in ( A ), to PSEx-coated plates. N.D. not detected. (*) Significant differences (Student’s t-test, P

    Article Snippet: All membranes were then incubated for 1 h at 37°C in a PBS solution of 0.1% (w/v) BSA and 0.05% (v/v) Tween-20 containing HRP-streptavidin (1:5,000—Sigma).

    Techniques: Labeling, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay

    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + streptavidin-Atto 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.

    Journal: Langmuir

    Article Title: From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups

    doi: 10.1021/acs.langmuir.7b01255

    Figure Lengend Snippet: Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + streptavidin-Atto 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.

    Article Snippet: Streptavidin-Atto 488 and streptavidin-Atto 655 were obtained from Sigma-Aldrich (Poole, UK).

    Techniques:

    Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + streptavidin-Atto 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.

    Journal: Langmuir

    Article Title: From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups

    doi: 10.1021/acs.langmuir.7b01255

    Figure Lengend Snippet: Stepwise assembly of multiple component protein nanopatterns using near-field lithography. (a) GFP + streptavidin-Atto 655. (b) IgG-FITC + streptavidin-Atto 655 + streptavidin-Atto 488 (c) IgG-FITC + streptavidin-Atto 655 + streptavidin-Alexa Fluor 488 + streptavidin-Alexa Fluor 750. A representative line section is provided beneath each micrograph, measured between the white arrowheads marked on each.

    Article Snippet: Streptavidin-Atto 488 and streptavidin-Atto 655 were obtained from Sigma-Aldrich (Poole, UK).

    Techniques:

    HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto 633-Streptavidin (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.

    Journal: Frontiers in Chemistry

    Article Title: Selective Targeting of Proteins by Hybrid Polyoxometalates: Interaction Between a Bis-Biotinylated Hybrid Conjugate and Avidin

    doi: 10.3389/fchem.2018.00278

    Figure Lengend Snippet: HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH 2 (Left) Na-POM-biot 2 (Right) , and stained with Atto 633-Streptavidin (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.

    Article Snippet: After incubation the cells were washed 3 times with PBS, fixed with paraformaldehyde, permeabilized by Triton and then stained with Atto 633-Streptavidin (Sigma #00336).

    Techniques: Incubation, Staining, Microscopy, Confocal Laser Scanning Microscopy, Concentration Assay

    Kinetics of inhibition of biotinylated D336C channels by streptavidin. (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.

    Journal: The Journal of General Physiology

    Article Title: Constraints on Voltage Sensor Movement in the Shaker K+ Channel

    doi: 10.1085/jgp.200609624

    Figure Lengend Snippet: Kinetics of inhibition of biotinylated D336C channels by streptavidin. (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.

    Article Snippet: Tetrameric streptavidin (Calbiochem/EMD Biosciences) was added by pipetting a 1 mg/ml (in ND-96) solution into the chamber and rapidly mixing to a final concentration of 200 μg/ml (3.3 μM).

    Techniques: Inhibition, Mass Spectrometry, Generated

    ( a ) Library of non- and biotinylated discotics; ( b ) Self-assembling multivalency of the discotics and the streptavidin mutants used in this study.

    Journal: International Journal of Molecular Sciences

    Article Title: Multivalent Protein Assembly Using Monovalent Self-Assembling Building Blocks

    doi: 10.3390/ijms141021189

    Figure Lengend Snippet: ( a ) Library of non- and biotinylated discotics; ( b ) Self-assembling multivalency of the discotics and the streptavidin mutants used in this study.

    Article Snippet: The concentration of monovalent and tetravalent streptavidin (purchased from Sigma, Zwijndrecht, The Netherlands) in PBS was adjusted to 3 mg/mL (determined by NanoDrop, Thermo Scientific, Wilmington, DE, USA); mass: 55 kDa, extinction coefficient: 200.000 M−1 cm−1 ).

    Techniques:

    ( a ) Emission spectra of the titration of monovalent Cy3-labeled streptavidin (Cy3-mSA, 0 nM–270 nM) to 1Biotin-Disc (1 μM). Inset: Emission spectra of the titration of monovalent Cy3-labeled streptavidin (0 nM–270 nM) to Inert-Disc (1 μM); ( b ) Normalized decrease in disc intensity at 525 nm upon addition of Cy3-mSA or Cy3-tSA to 1Biotin-Disc and Inert-Disc (both 1 μM); Dashed line represents the intensity, where half of the donor intensity is quenched; ( c ) Photography of cuvettes under UV-light three days after Förster resonance energy transfer (FRET) titration.

    Journal: International Journal of Molecular Sciences

    Article Title: Multivalent Protein Assembly Using Monovalent Self-Assembling Building Blocks

    doi: 10.3390/ijms141021189

    Figure Lengend Snippet: ( a ) Emission spectra of the titration of monovalent Cy3-labeled streptavidin (Cy3-mSA, 0 nM–270 nM) to 1Biotin-Disc (1 μM). Inset: Emission spectra of the titration of monovalent Cy3-labeled streptavidin (0 nM–270 nM) to Inert-Disc (1 μM); ( b ) Normalized decrease in disc intensity at 525 nm upon addition of Cy3-mSA or Cy3-tSA to 1Biotin-Disc and Inert-Disc (both 1 μM); Dashed line represents the intensity, where half of the donor intensity is quenched; ( c ) Photography of cuvettes under UV-light three days after Förster resonance energy transfer (FRET) titration.

    Article Snippet: The concentration of monovalent and tetravalent streptavidin (purchased from Sigma, Zwijndrecht, The Netherlands) in PBS was adjusted to 3 mg/mL (determined by NanoDrop, Thermo Scientific, Wilmington, DE, USA); mass: 55 kDa, extinction coefficient: 200.000 M−1 cm−1 ).

    Techniques: Titration, Labeling, Förster Resonance Energy Transfer

    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with streptavidin-HRP and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input

    Journal: Journal of Inflammation (London, England)

    Article Title: Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) regulates inflammasomes

    doi: 10.1186/1476-9255-7-23

    Figure Lengend Snippet: ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with streptavidin-HRP and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input".

    Article Snippet: Bound proteins were washed 4 times in HKMEN buffer supplemented with protease inhibitors, boiled in Laemmli buffer, separated by SDS/PAGE, transferred onto a PVDF membrane, and detected with Streptavidin-HRP in conjugation with an enhanced chemiluminescent reagent (Millipore).

    Techniques: Incubation, In Vitro

    Phagocytosis Inhibits 3D Migration (A) Marrow macrophages were engineered and mixed with streptavidin beads of 900 nm to 6.7 μm diameter or else with disaggregated tdTom A549 tumors. Beads are opsonized with anti-streptavidin except for a non-targeted control in which SIRPα-inhibited macrophages were incubated with 2.1 μm lacking anti-streptavidin. These were not engulfed. After pre-incubation of the mixtures for up to 150 min, mixtures were plated for 24 hr on top of 5-μm pore transwells. Cells collected from transwell tops and bottoms were stained for CD11b + ,F4/80 + and analyzed by flow cytometry for engulfment (STAR Methods). (B) Bottom/total cell counts gives the %-migrating macrophages, with the non-opsonized control bead result at 0 (n = 3). (C) The %-Eating indicates internalization of at least one bead per macrophages on top of trans-wells (or else just after the pre-incubation) (n = 3). (D) The average number of opsonized beads engulfed per macrophage is ~2 for all bead sizes (n = 3). ). A’PB macrophages were mixed at 10:1 ratio with disaggregated tumor cells or tissue cells. Nearly all A’PB macrophages become tdTom + and rarely migrate, whereas ~50-fold more of the same A’PB macrophages successfully migrate through the pores when incubated with control lung tissue (n = 3). Error bars in (B)–(E) represent mean + SEM.

    Journal: Current biology : CB

    Article Title: SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors

    doi: 10.1016/j.cub.2017.06.005

    Figure Lengend Snippet: Phagocytosis Inhibits 3D Migration (A) Marrow macrophages were engineered and mixed with streptavidin beads of 900 nm to 6.7 μm diameter or else with disaggregated tdTom A549 tumors. Beads are opsonized with anti-streptavidin except for a non-targeted control in which SIRPα-inhibited macrophages were incubated with 2.1 μm lacking anti-streptavidin. These were not engulfed. After pre-incubation of the mixtures for up to 150 min, mixtures were plated for 24 hr on top of 5-μm pore transwells. Cells collected from transwell tops and bottoms were stained for CD11b + ,F4/80 + and analyzed by flow cytometry for engulfment (STAR Methods). (B) Bottom/total cell counts gives the %-migrating macrophages, with the non-opsonized control bead result at 0 (n = 3). (C) The %-Eating indicates internalization of at least one bead per macrophages on top of trans-wells (or else just after the pre-incubation) (n = 3). (D) The average number of opsonized beads engulfed per macrophage is ~2 for all bead sizes (n = 3). ). A’PB macrophages were mixed at 10:1 ratio with disaggregated tumor cells or tissue cells. Nearly all A’PB macrophages become tdTom + and rarely migrate, whereas ~50-fold more of the same A’PB macrophages successfully migrate through the pores when incubated with control lung tissue (n = 3). Error bars in (B)–(E) represent mean + SEM.

    Article Snippet: Beads were opsonized with anti-streptavidin antibody (S6390, Sigma).

    Techniques: Migration, Incubation, Staining, Flow Cytometry, Cytometry