amylose resin  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Amylose Resin
    Description:
    Amylose Resin 100 ml
    Catalog Number:
    e8021l
    Price:
    1158
    Size:
    100 ml
    Category:
    Protein Purification Kit Components
    Buy from Supplier


    Structured Review

    New England Biolabs amylose resin
    Amylose Resin
    Amylose Resin 100 ml
    https://www.bioz.com/result/amylose resin/product/New England Biolabs
    Average 99 stars, based on 974 article reviews
    Price from $9.99 to $1999.99
    amylose resin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    Related Articles

    Affinity Chromatography:

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

    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.

    Mutagenesis:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

    Construct:

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

    Purification:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

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

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase
    Article Snippet: .. 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: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    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. .. 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–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES
    Article Snippet: .. MBP was expressed in the same manner as other proteins and purified by amylose resin (NEB) in TBS150 containing 1 mM EDTA by using elution buffer (20 mM maltose and 1 mM EDTA in TBS150). ..

    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: .. 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.

    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.

    Recombinant:

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

    Lysis:

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

    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: .. 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.

    Plasmid Preparation:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs amylose resin based affinity chromatography
    SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach <t>(amylose</t> <t>resin-based</t> <t>affinity</t> <t>chromatography</t> 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.
    Amylose Resin Based Affinity Chromatography, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amylose resin based affinity chromatography/product/New England Biolabs
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    amylose resin based affinity chromatography - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    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.

    Journal: International journal for parasitology

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

    doi: 10.1016/j.ijpara.2006.10.014

    Figure Lengend 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.

    Article Snippet: 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).

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

    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

    Calmodulin binds to the kinase domain of SRK in vitro. A, Binding of GST or the GST protein fused with either wild-type or mutant forms of the SRK 29 kinase domain to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. B, Binding of the integral SRK protein to calmodulin-Sepharose beads. A recombinant hexa-His epitope-tagged, kinase-inactive form of SRK ( m SRK 3 ). C, Binding of Brassica calmodulin to glutathione-Sepharose beads carrying either GST or GST protein fused to either wild-type or mutant forms of the SRK 29 kinase domain. Eluted proteins were detected by silver staining. Note that calmodulin alters its conformation in the presence of calcium resulting in a change in mobility. CAM, Calmodulin with no bound Ca 2+ ; CAM/Ca, calmodulin with bound Ca 2+ ; C, purified fusion protein; UB, unbound fraction; W1 and W40, washes; E1 to E4, eluted fractions. C and UB, One-quarter of the amount of protein loaded on and unbound to the column, respectively.

    Journal: Plant Physiology

    Article Title: Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

    doi: 10.1104/pp.103.023846

    Figure Lengend Snippet: Calmodulin binds to the kinase domain of SRK in vitro. A, Binding of GST or the GST protein fused with either wild-type or mutant forms of the SRK 29 kinase domain to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. B, Binding of the integral SRK protein to calmodulin-Sepharose beads. A recombinant hexa-His epitope-tagged, kinase-inactive form of SRK ( m SRK 3 ). C, Binding of Brassica calmodulin to glutathione-Sepharose beads carrying either GST or GST protein fused to either wild-type or mutant forms of the SRK 29 kinase domain. Eluted proteins were detected by silver staining. Note that calmodulin alters its conformation in the presence of calcium resulting in a change in mobility. CAM, Calmodulin with no bound Ca 2+ ; CAM/Ca, calmodulin with bound Ca 2+ ; C, purified fusion protein; UB, unbound fraction; W1 and W40, washes; E1 to E4, eluted fractions. C and UB, One-quarter of the amount of protein loaded on and unbound to the column, respectively.

    Article Snippet: Binding to SNX1 was detected by incubating E. coli extracts containing equivalent amounts of soluble, recombinant GST- or MBP-kinase domain fusion proteins in affinity lysis buffer (50 m m HEPES [pH 7.4], 150 m m NaCl, 10 m m EDTA, 1 m m DTT, and antiprotease cocktail) with 25 μL of glutathione-Sepharose (Amersham Pharmacia Biotech Inc., Piscataway, NJ) or amylose-Sepharose resin (New England BioLabs, Beverly, MA), respectively.

    Techniques: In Vitro, Binding Assay, Mutagenesis, Recombinant, Silver Staining, Chick Chorioallantoic Membrane Assay, Purification

    Calmodulin binds to sub-domain VIa of the kinase domain of SRK in vitro. A, Amino acid sequence of the HEL1 region of sub-domain VIa of SRK 3 . The wheel projection shows the amphiphilic nature of HEL1. The sequence of the control peptide PEP1 is also shown. B, Binding of GST::HEL1 and GST::PEP1 fusion proteins to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. C, purified GST fusion protein; UB, unbound fraction; W1 and W40, first and last washes; E1 to E4, eluted fractions.

    Journal: Plant Physiology

    Article Title: Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

    doi: 10.1104/pp.103.023846

    Figure Lengend Snippet: Calmodulin binds to sub-domain VIa of the kinase domain of SRK in vitro. A, Amino acid sequence of the HEL1 region of sub-domain VIa of SRK 3 . The wheel projection shows the amphiphilic nature of HEL1. The sequence of the control peptide PEP1 is also shown. B, Binding of GST::HEL1 and GST::PEP1 fusion proteins to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. C, purified GST fusion protein; UB, unbound fraction; W1 and W40, first and last washes; E1 to E4, eluted fractions.

    Article Snippet: Binding to SNX1 was detected by incubating E. coli extracts containing equivalent amounts of soluble, recombinant GST- or MBP-kinase domain fusion proteins in affinity lysis buffer (50 m m HEPES [pH 7.4], 150 m m NaCl, 10 m m EDTA, 1 m m DTT, and antiprotease cocktail) with 25 μL of glutathione-Sepharose (Amersham Pharmacia Biotech Inc., Piscataway, NJ) or amylose-Sepharose resin (New England BioLabs, Beverly, MA), respectively.

    Techniques: In Vitro, Sequencing, Binding Assay, Purification

    Calmodulin binds to the kinase domains of SFR1, RLK4, and CLV1, but not BRI1, in vitro. A, Analysis of the binding of the kinase domains of SFR1, RLK4, CLV1, and BRI1 to calmodulin-Sepharose. The kinase domains were expressed as fusion proteins with either GST or MBP. Both a wild-type (GST::SFR1kin) and a kinase-inactive mutant form (GST::SFR1kin K555R ) of the SFR1 kinase domain were tested. GST- and MBP-containing proteins were detected with anti-GST and anti-MBP antibodies, respectively. B, Abundance of calmodulin transcripts in different Brassica organs. Calmodulin transcripts were detected with a probe corresponding to the entire coding sequence. Lower, Ethidium bromide-stained rRNA. The positions of RNA size markers are shown at right in kilobase pairs. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma.

    Journal: Plant Physiology

    Article Title: Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

    doi: 10.1104/pp.103.023846

    Figure Lengend Snippet: Calmodulin binds to the kinase domains of SFR1, RLK4, and CLV1, but not BRI1, in vitro. A, Analysis of the binding of the kinase domains of SFR1, RLK4, CLV1, and BRI1 to calmodulin-Sepharose. The kinase domains were expressed as fusion proteins with either GST or MBP. Both a wild-type (GST::SFR1kin) and a kinase-inactive mutant form (GST::SFR1kin K555R ) of the SFR1 kinase domain were tested. GST- and MBP-containing proteins were detected with anti-GST and anti-MBP antibodies, respectively. B, Abundance of calmodulin transcripts in different Brassica organs. Calmodulin transcripts were detected with a probe corresponding to the entire coding sequence. Lower, Ethidium bromide-stained rRNA. The positions of RNA size markers are shown at right in kilobase pairs. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma.

    Article Snippet: Binding to SNX1 was detected by incubating E. coli extracts containing equivalent amounts of soluble, recombinant GST- or MBP-kinase domain fusion proteins in affinity lysis buffer (50 m m HEPES [pH 7.4], 150 m m NaCl, 10 m m EDTA, 1 m m DTT, and antiprotease cocktail) with 25 μL of glutathione-Sepharose (Amersham Pharmacia Biotech Inc., Piscataway, NJ) or amylose-Sepharose resin (New England BioLabs, Beverly, MA), respectively.

    Techniques: In Vitro, Binding Assay, Mutagenesis, Sequencing, Staining

    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