recombinant mbp mcp conjugated amylose resin  (New England Biolabs)


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    Amylose Resin
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    Amylose Resin 100 ml
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    E8021L
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    Protein Purification Kit Components
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    New England Biolabs recombinant mbp mcp conjugated amylose resin
    Amylose Resin
    Amylose Resin 100 ml
    https://www.bioz.com/result/recombinant mbp mcp conjugated amylose resin/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    recombinant mbp mcp conjugated amylose resin - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1"

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1

    Journal: Oncogenesis

    doi: 10.1038/s41389-019-0182-7

    Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.
    Figure Legend Snippet: Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.

    Techniques Used: Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Transfection, Mutagenesis

    Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.
    Figure Legend Snippet: Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Recombinant, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

    2) Product Images from "Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation"

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201609073

    Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.
    Figure Legend Snippet: Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Immunofluorescence, Staining, Construct, Functional Assay, Mutagenesis

    3) Product Images from "Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein"

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008381

    On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.
    Figure Legend Snippet: On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Techniques Used: Copurification, Labeling, SDS Page, Staining, Fluorescence, Expressing, Incubation, Purification

    Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.
    Figure Legend Snippet: Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Techniques Used: Labeling, Binding Assay, Purification, Sequencing, Incubation, SDS Page, Staining, Western Blot, Fluorescence, Affinity Chromatography

    4) Product Images from "An unfolded protein-induced conformational switch activates mammalian IRE1"

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1

    Journal: eLife

    doi: 10.7554/eLife.30700

    hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p
    Figure Legend Snippet: hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Techniques Used: Binding Assay

    5) Product Images from "Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase"

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase

    Journal: International journal for parasitology

    doi: 10.1016/j.ijpara.2006.10.014

    SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.
    Figure Legend Snippet: SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.

    Techniques Used: SDS Page, Binding Assay, Purification, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Gel Extraction, Marker, Gel Purification, Affinity Purification

    Related Articles

    Purification:

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase
    Article Snippet: Insoluble debris was removed by centrifugation. .. The MBP-CpPKS1-AL-ACP fusion protein was purified using amylose resin-based affinity chromatography according to the manufacturer’s standard protocol (New England Biolabs). .. SDS-PAGE analysis of the purified protein revealed two distinct bands; one corresponding to full-length MBP-fused CpPKS1-AL-ACP (~138-kDa) and another at approximately 80-kDa.

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: For purification, cells were resuspended in Lysis Buffer (50 mM HEPES pH 7.2, 400 mM NaCl, 4 mM dithiothreitol (DTT)(or 5 mM β-mercaptoethanol, if a nickel column was used)) and were lysed in an Avestin EmulsiFlex-C3 cell disruptor at 16,000 psi. .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: His-MBP-Kindlin-2 was expressed in E. coli BL21 and purified with His-Select HF Nickel Affinity Gel (Sigma-Aldrich). .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Affinity Chromatography:

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase
    Article Snippet: Insoluble debris was removed by centrifugation. .. The MBP-CpPKS1-AL-ACP fusion protein was purified using amylose resin-based affinity chromatography according to the manufacturer’s standard protocol (New England Biolabs). .. SDS-PAGE analysis of the purified protein revealed two distinct bands; one corresponding to full-length MBP-fused CpPKS1-AL-ACP (~138-kDa) and another at approximately 80-kDa.

    Recombinant:

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1
    Article Snippet: miRNA and protein sequencing after MS2 pulldown assays MS2 pulldown assays were performed as previously described using a recombinant fusion protein (MBP-MCP) that contains a maltose-binding domain (MBP) and a domain (MCP) that recognizes the MS2 hairpins . .. Briefly, recombinant MBP-MCP-conjugated amylose resin (NEB, USA) was prepared at 4 °C. .. Cell lysates prepared from cells expressing MS2-tagged MACC1-AS1 or MACC1-AS1 mutants were incubated with MBP-MCP-coated amylose resin at 4 °C for 4 h in the presence of RNase and protease inhibitors.

    Construct:

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: For purification, cells were resuspended in Lysis Buffer (50 mM HEPES pH 7.2, 400 mM NaCl, 4 mM dithiothreitol (DTT)(or 5 mM β-mercaptoethanol, if a nickel column was used)) and were lysed in an Avestin EmulsiFlex-C3 cell disruptor at 16,000 psi. .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Lysis:

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: For purification, cells were resuspended in Lysis Buffer (50 mM HEPES pH 7.2, 400 mM NaCl, 4 mM dithiothreitol (DTT)(or 5 mM β-mercaptoethanol, if a nickel column was used)) and were lysed in an Avestin EmulsiFlex-C3 cell disruptor at 16,000 psi. .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Activity Assay:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Fluorescence:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Binding Assay:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: His-MBP-Kindlin-2 was expressed in E. coli BL21 and purified with His-Select HF Nickel Affinity Gel (Sigma-Aldrich). .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Incubation:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: His-MBP-Kindlin-2 was expressed in E. coli BL21 and purified with His-Select HF Nickel Affinity Gel (Sigma-Aldrich). .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Article Title: Structure of a mitochondrial fission dynamin in the closed conformation
    Article Snippet: .. The supernatant was incubated with amylose resin (New England BioLabs) for 3 hrs at 4 °C with gentle stirring, and loaded into a gravity column. ..

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    New England Biolabs recombinant mbp mcp conjugated amylose resin
    Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through <t>MBP-MCP.</t> d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.
    Recombinant Mbp Mcp Conjugated Amylose Resin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.

    Journal: Oncogenesis

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1

    doi: 10.1038/s41389-019-0182-7

    Figure Lengend Snippet: Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.

    Article Snippet: Briefly, recombinant MBP-MCP-conjugated amylose resin (NEB, USA) was prepared at 4 °C.

    Techniques: Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Transfection, Mutagenesis

    Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.

    Journal: Oncogenesis

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1

    doi: 10.1038/s41389-019-0182-7

    Figure Lengend Snippet: Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.

    Article Snippet: Briefly, recombinant MBP-MCP-conjugated amylose resin (NEB, USA) was prepared at 4 °C.

    Techniques: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Recombinant, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

    Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Journal: The Journal of Cell Biology

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    doi: 10.1083/jcb.201609073

    Figure Lengend Snippet: Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Article Snippet: To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation.

    Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Immunofluorescence, Staining, Construct, Functional Assay, Mutagenesis

    On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Journal: PLoS ONE

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    doi: 10.1371/journal.pone.0008381

    Figure Lengend Snippet: On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Article Snippet: Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein.

    Techniques: Copurification, Labeling, SDS Page, Staining, Fluorescence, Expressing, Incubation, Purification

    Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Journal: PLoS ONE

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    doi: 10.1371/journal.pone.0008381

    Figure Lengend Snippet: Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Article Snippet: Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein.

    Techniques: Labeling, Binding Assay, Purification, Sequencing, Incubation, SDS Page, Staining, Western Blot, Fluorescence, Affinity Chromatography

    hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Journal: eLife

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1

    doi: 10.7554/eLife.30700

    Figure Lengend Snippet: hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Article Snippet: MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer.

    Techniques: Binding Assay