streptavidin agarose mini column  (Millipore)


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    Millipore streptavidin agarose mini column
    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by <t>streptavidin-agarose</t> and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.
    Streptavidin Agarose Mini Column, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose mini column/product/Millipore
    Average 93 stars, based on 1 article reviews
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    streptavidin agarose mini column - by Bioz Stars, 2020-04
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    biotinylated protein

    Images

    1) Product Images from "The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity"

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17449

    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.
    Figure Legend Snippet: Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.

    Techniques Used: SDS Page, Incubation, Western Blot

    2) Product Images from "The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity"

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17449

    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.
    Figure Legend Snippet: Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.

    Techniques Used: SDS Page, Incubation, Western Blot

    Related Articles

    Blocking Assay:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: In brief, the VPAC1-CHO and VPAC1-Cys37/Ala-CHO cell lysates were incubated overnight with 1M NEM (Sigma, USA) at 4°C to block all the free sulfhydryls. .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Produced:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: And 4M biotin-HPDP (ProteoChem, USA) was added later to react with the free sulfhydryls produced by HA to achieve the biotin exchange labeling of the palmitoylation. .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Incubation:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: In brief, the VPAC1-CHO and VPAC1-Cys37/Ala-CHO cell lysates were incubated overnight with 1M NEM (Sigma, USA) at 4°C to block all the free sulfhydryls. .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Labeling:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: In brief, after the metabolic labeling the palmitoylated proteins with palmitate orthologs alkyne-linked 17-Odya (Cayman Chemical, USA), the cells lysates were further processed through the click reaction between biotin-azide (Cayman Chemical, USA) and alkyne-linked 17-Odya, which make the palmitoylated proteins covalently cross-linked with biotin at the S-palmitoylation sites. .. The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: And 4M biotin-HPDP (ProteoChem, USA) was added later to react with the free sulfhydryls produced by HA to achieve the biotin exchange labeling of the palmitoylation. .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Western Blot:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: .. The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China). ..

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China). ..

    SDS Page:

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity
    Article Snippet: .. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China). ..

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    Millipore streptavidin sepharose affinity columns
    <t>Streptavidin-affinity-enriched</t> PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using <t>streptavidin—Sepharose</t> affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.
    Streptavidin Sepharose Affinity Columns, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin sepharose affinity columns/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin sepharose affinity columns - by Bioz Stars, 2020-04
    88/100 stars
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    Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Journal: PLoS ONE

    Article Title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    doi: 10.1371/journal.pone.0133033

    Figure Lengend Snippet: Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Article Snippet: Protein fractions were eluted by streptavidin—Sepharose affinity columns, desalted, concentrated using AmiconTM Ultrafree centrifugal filters (Millipore), and visualized using a FOCUS-FAST silver-stain kit.

    Techniques: Affinity Chromatography, Far Western Blot, Silver Staining