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  • 99
    Millipore monoclonal anti flag m2 antibody
    CAF-1 subunits interact with <t>Cse4.</t> Whole cell extracts were used to carry out co-IPs in ( A – C ). (A) Left panel: strains carrying GFP-tagged chaperones, Scm3, Vps75, Nap1 and Cac2 subunit of CAF-1 were used in co-IP. Only Cac2-GFP pulled down Cse4. A control IP was performed from a strain lacking the GFP tag. Scm3-GFP IP was the positive control. Right panel: affinity-tagged strains <t>(Cse4-Myc/Cac2-FLAG</t> and Cse4-Myc/Hir1-FLAG) were used for co-IP. A control IP was performed from a strain lacking the FLAG tag. In anti-FLAG co-IP, Hir1-FLAG did not pull down Cse4, only Cac2-FLAG pulled down Cse4-Myc. (B) Cac1 and Cac3 subunits of CAF-1 are required for interaction with Cse4. Upper panel: WT or cac2Δ strains carrying FLAG-tagged Cac1 or Cac3 were used for co-IP. A strain lacking the tag on Cac1 and Cac3 was used as the negative control. In anti-FLAG co-IP, both Cac1 and Cac3 pulled down noticeably more Cse4-Myc. These interactions became weaker with CAC2 deletion. Lower panel: WT, cac1Δ or cac3Δ strains carrying FLAG-tagged Cac2 were used in anti-FLAG co-IP. A strain lacking the tag was used as the control. Cac2/Cse4 interaction was completely abolished in cac1Δ or cac3Δ strains. (C) CAF-1 can interact with Cse4 outside of S phase. Affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Scm3-FLAG) were used in anti-FLAG co-IP using asynchronously grown cells and G2/M-arrested cells. A strain lacking the FLAG tag served as a negative control. Scm3 was used as a positive control. Cac2 interacted with Cse4 in both asynchronously grown cells as well as G2/M-arrested cells. A non-specific band is marked with an asterisk.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 42777 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2020-11
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    99
    Millipore monoclonal anti flag m2 peroxidase hrp antibody
    CAF-1 subunits interact with <t>Cse4.</t> Whole cell extracts were used to carry out co-IPs in ( A – C ). (A) Left panel: strains carrying GFP-tagged chaperones, Scm3, Vps75, Nap1 and Cac2 subunit of CAF-1 were used in co-IP. Only Cac2-GFP pulled down Cse4. A control IP was performed from a strain lacking the GFP tag. Scm3-GFP IP was the positive control. Right panel: affinity-tagged strains <t>(Cse4-Myc/Cac2-FLAG</t> and Cse4-Myc/Hir1-FLAG) were used for co-IP. A control IP was performed from a strain lacking the FLAG tag. In anti-FLAG co-IP, Hir1-FLAG did not pull down Cse4, only Cac2-FLAG pulled down Cse4-Myc. (B) Cac1 and Cac3 subunits of CAF-1 are required for interaction with Cse4. Upper panel: WT or cac2Δ strains carrying FLAG-tagged Cac1 or Cac3 were used for co-IP. A strain lacking the tag on Cac1 and Cac3 was used as the negative control. In anti-FLAG co-IP, both Cac1 and Cac3 pulled down noticeably more Cse4-Myc. These interactions became weaker with CAC2 deletion. Lower panel: WT, cac1Δ or cac3Δ strains carrying FLAG-tagged Cac2 were used in anti-FLAG co-IP. A strain lacking the tag was used as the control. Cac2/Cse4 interaction was completely abolished in cac1Δ or cac3Δ strains. (C) CAF-1 can interact with Cse4 outside of S phase. Affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Scm3-FLAG) were used in anti-FLAG co-IP using asynchronously grown cells and G2/M-arrested cells. A strain lacking the FLAG tag served as a negative control. Scm3 was used as a positive control. Cac2 interacted with Cse4 in both asynchronously grown cells as well as G2/M-arrested cells. A non-specific band is marked with an asterisk.
    Monoclonal Anti Flag M2 Peroxidase Hrp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 peroxidase hrp antibody/product/Millipore
    Average 99 stars, based on 4318 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 peroxidase hrp antibody - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

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    CAF-1 subunits interact with Cse4. Whole cell extracts were used to carry out co-IPs in ( A – C ). (A) Left panel: strains carrying GFP-tagged chaperones, Scm3, Vps75, Nap1 and Cac2 subunit of CAF-1 were used in co-IP. Only Cac2-GFP pulled down Cse4. A control IP was performed from a strain lacking the GFP tag. Scm3-GFP IP was the positive control. Right panel: affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Hir1-FLAG) were used for co-IP. A control IP was performed from a strain lacking the FLAG tag. In anti-FLAG co-IP, Hir1-FLAG did not pull down Cse4, only Cac2-FLAG pulled down Cse4-Myc. (B) Cac1 and Cac3 subunits of CAF-1 are required for interaction with Cse4. Upper panel: WT or cac2Δ strains carrying FLAG-tagged Cac1 or Cac3 were used for co-IP. A strain lacking the tag on Cac1 and Cac3 was used as the negative control. In anti-FLAG co-IP, both Cac1 and Cac3 pulled down noticeably more Cse4-Myc. These interactions became weaker with CAC2 deletion. Lower panel: WT, cac1Δ or cac3Δ strains carrying FLAG-tagged Cac2 were used in anti-FLAG co-IP. A strain lacking the tag was used as the control. Cac2/Cse4 interaction was completely abolished in cac1Δ or cac3Δ strains. (C) CAF-1 can interact with Cse4 outside of S phase. Affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Scm3-FLAG) were used in anti-FLAG co-IP using asynchronously grown cells and G2/M-arrested cells. A strain lacking the FLAG tag served as a negative control. Scm3 was used as a positive control. Cac2 interacted with Cse4 in both asynchronously grown cells as well as G2/M-arrested cells. A non-specific band is marked with an asterisk.

    Journal: Nucleic Acids Research

    Article Title: Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast

    doi: 10.1093/nar/gky169

    Figure Lengend Snippet: CAF-1 subunits interact with Cse4. Whole cell extracts were used to carry out co-IPs in ( A – C ). (A) Left panel: strains carrying GFP-tagged chaperones, Scm3, Vps75, Nap1 and Cac2 subunit of CAF-1 were used in co-IP. Only Cac2-GFP pulled down Cse4. A control IP was performed from a strain lacking the GFP tag. Scm3-GFP IP was the positive control. Right panel: affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Hir1-FLAG) were used for co-IP. A control IP was performed from a strain lacking the FLAG tag. In anti-FLAG co-IP, Hir1-FLAG did not pull down Cse4, only Cac2-FLAG pulled down Cse4-Myc. (B) Cac1 and Cac3 subunits of CAF-1 are required for interaction with Cse4. Upper panel: WT or cac2Δ strains carrying FLAG-tagged Cac1 or Cac3 were used for co-IP. A strain lacking the tag on Cac1 and Cac3 was used as the negative control. In anti-FLAG co-IP, both Cac1 and Cac3 pulled down noticeably more Cse4-Myc. These interactions became weaker with CAC2 deletion. Lower panel: WT, cac1Δ or cac3Δ strains carrying FLAG-tagged Cac2 were used in anti-FLAG co-IP. A strain lacking the tag was used as the control. Cac2/Cse4 interaction was completely abolished in cac1Δ or cac3Δ strains. (C) CAF-1 can interact with Cse4 outside of S phase. Affinity-tagged strains (Cse4-Myc/Cac2-FLAG and Cse4-Myc/Scm3-FLAG) were used in anti-FLAG co-IP using asynchronously grown cells and G2/M-arrested cells. A strain lacking the FLAG tag served as a negative control. Scm3 was used as a positive control. Cac2 interacted with Cse4 in both asynchronously grown cells as well as G2/M-arrested cells. A non-specific band is marked with an asterisk.

    Article Snippet: Antibodies used for ChIP experiments are 2 μl of α-Cse4 antibody (polyclonal rabbit antibody against recombinant Cse4) and anti-FLAG (Sigma-F3165).

    Techniques: Co-Immunoprecipitation Assay, Positive Control, FLAG-tag, Negative Control

    When Scm3 is absent and Cse4 levels are high, CAF-1 may help target Cse4 to the centromere. ( A ) Cac1 localization to centromeres is cell-cycle independent. ChIP was performed for Cac1 using a FLAG-tagged strain using asynchronously growing cells and also G1, S and G2/M arrested cells. qPCR was used to detect Cac1 levels at CEN3. The y -axis indicates arbitrary units representing the Cac1 enrichment at CEN3 from ChIP performed with and without antibody, with respect to the signal for total chromatin for each sample. The error bars represent the standard deviation for ChIP performed in triplicate. ( B ) Rescue of Scm3 off strain by overexpression of Cse4 is less efficient when CAC2 is deleted. Strains containing a single copy of Scm3 under the control of the gal promoter, allowing the expression of Scm3 to be controlled with glucose/galactose, were used in the spotting assay. Cse4 was under the control of a copper-inducible promoter on a plasmid. ‘EV’ indicates empty vector. When Scm3 is expressed (Scm3 on ), no growth differences are observed in 10-fold serial dilutions. Induction of Cse4 does not efficiently rescue growth of the Scm3 off strain when CAC2 is deleted as compared to WT. Three independent isolates of the cac2Δ strain (1, 2 and 3) were tested.

    Journal: Nucleic Acids Research

    Article Title: Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast

    doi: 10.1093/nar/gky169

    Figure Lengend Snippet: When Scm3 is absent and Cse4 levels are high, CAF-1 may help target Cse4 to the centromere. ( A ) Cac1 localization to centromeres is cell-cycle independent. ChIP was performed for Cac1 using a FLAG-tagged strain using asynchronously growing cells and also G1, S and G2/M arrested cells. qPCR was used to detect Cac1 levels at CEN3. The y -axis indicates arbitrary units representing the Cac1 enrichment at CEN3 from ChIP performed with and without antibody, with respect to the signal for total chromatin for each sample. The error bars represent the standard deviation for ChIP performed in triplicate. ( B ) Rescue of Scm3 off strain by overexpression of Cse4 is less efficient when CAC2 is deleted. Strains containing a single copy of Scm3 under the control of the gal promoter, allowing the expression of Scm3 to be controlled with glucose/galactose, were used in the spotting assay. Cse4 was under the control of a copper-inducible promoter on a plasmid. ‘EV’ indicates empty vector. When Scm3 is expressed (Scm3 on ), no growth differences are observed in 10-fold serial dilutions. Induction of Cse4 does not efficiently rescue growth of the Scm3 off strain when CAC2 is deleted as compared to WT. Three independent isolates of the cac2Δ strain (1, 2 and 3) were tested.

    Article Snippet: Antibodies used for ChIP experiments are 2 μl of α-Cse4 antibody (polyclonal rabbit antibody against recombinant Cse4) and anti-FLAG (Sigma-F3165).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation, Over Expression, Expressing, Spotting Assay, Plasmid Preparation

    Analysis of scFv-G4 expression and its specificity. (A) Detection of scFv-G4 expression. Total protein from scFv transgenic Sf9-III cells was extracted and analyzed by SDS-PAGE. A FLAG mAb was used to detect the expression of scFv-G4. (B) Verification of scFv-G4 specificity. Spore protein were analyzed by SDS-PAGE. ScFv-G4 transgenic Sf9-III and negative control Sf9-III cell lysate solutions were used as the primary antibody, after which FLAG mAb or polyclonal antibodies were used as the secondary antibodies and then HRP-labeled goat IgG was used as the third antibody. M: protein marker (Transgene, China).

    Journal: PLoS ONE

    Article Title: Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells

    doi: 10.1371/journal.pone.0193065

    Figure Lengend Snippet: Analysis of scFv-G4 expression and its specificity. (A) Detection of scFv-G4 expression. Total protein from scFv transgenic Sf9-III cells was extracted and analyzed by SDS-PAGE. A FLAG mAb was used to detect the expression of scFv-G4. (B) Verification of scFv-G4 specificity. Spore protein were analyzed by SDS-PAGE. ScFv-G4 transgenic Sf9-III and negative control Sf9-III cell lysate solutions were used as the primary antibody, after which FLAG mAb or polyclonal antibodies were used as the secondary antibodies and then HRP-labeled goat IgG was used as the third antibody. M: protein marker (Transgene, China).

    Article Snippet: As scFv lacks the constant regions and cannot be recognized by anti-mouse or anti-rabbit IgG antibodies, a mouse anti-FLAG mAb (Sigma, Saint Louis, Missouri, USA) and a rabbit polyclonal antibody (Rockland Immunochemical, Philadelphia, Pennsylvania, USA) were used as secondary antibodies for scFv-G4 recognition in western blot.

    Techniques: Expressing, Transgenic Assay, SDS Page, Negative Control, Labeling, Marker

    Assembly of complexes of Cdc37 and Hsp90 phosphomimetic variants with clients and cochaperones. a Binary complex formation between Cdc37 variants and bRaf (left), and between Hsp90β variants and Cdc37 (right), followed by ITC. The corresponding K d values are displayed in the inset. Error bars in the K d values correspond to the errors resulted in fitting of the data into a single binding site model. b HEK-293 cells were cotransfected with indicated HA-tagged Cdc37 and FLAG-tagged bRaf plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed and analyzed for Cdc37 interaction by SDS-PAGE/western blot, using anti-HA antibody. c HEK-293 cells were transfected with FLAG-tagged Hsp90, Hsp90 Y197E , or Hsp90 Y197F plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed three times before analysis by SDS-PAGE/western blot. Co-precipitating endogenous Hsp70, Aha1, p23, Hop, Fkbp59, and Cdk4 were detected with specific antibodies. d HEK-293 cells were transfected with the indicated Hsp90, androgen receptor (AR), and glucocorticoid receptor (GR) plasmids. Proteins were precipitated with GFP-Trap resin (left) or ANTI-FLAG M2 agarose (right) for 1 h at 4 °C with rotation. Bead pellets were washed three times with lysis buffer before analysis by SDS-PAGE/western blot as indicated. AR was visualized with anti-GFP antibody, GR was visualized with a specific antibody, and Hsp90 was visualized with anti-FLAG antibody

    Journal: Nature Communications

    Article Title: Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation

    doi: 10.1038/s41467-017-02711-w

    Figure Lengend Snippet: Assembly of complexes of Cdc37 and Hsp90 phosphomimetic variants with clients and cochaperones. a Binary complex formation between Cdc37 variants and bRaf (left), and between Hsp90β variants and Cdc37 (right), followed by ITC. The corresponding K d values are displayed in the inset. Error bars in the K d values correspond to the errors resulted in fitting of the data into a single binding site model. b HEK-293 cells were cotransfected with indicated HA-tagged Cdc37 and FLAG-tagged bRaf plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed and analyzed for Cdc37 interaction by SDS-PAGE/western blot, using anti-HA antibody. c HEK-293 cells were transfected with FLAG-tagged Hsp90, Hsp90 Y197E , or Hsp90 Y197F plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed three times before analysis by SDS-PAGE/western blot. Co-precipitating endogenous Hsp70, Aha1, p23, Hop, Fkbp59, and Cdk4 were detected with specific antibodies. d HEK-293 cells were transfected with the indicated Hsp90, androgen receptor (AR), and glucocorticoid receptor (GR) plasmids. Proteins were precipitated with GFP-Trap resin (left) or ANTI-FLAG M2 agarose (right) for 1 h at 4 °C with rotation. Bead pellets were washed three times with lysis buffer before analysis by SDS-PAGE/western blot as indicated. AR was visualized with anti-GFP antibody, GR was visualized with a specific antibody, and Hsp90 was visualized with anti-FLAG antibody

    Article Snippet: Antibody sources/clone #’s are as follows: GR (glucocorticoid receptor) monoclonal antibody is from Santa Cruz Biotechnology (cat # sc-393232, 1:5000); GFP monoclonal antibody is from Cell Signaling (cat #2956, 1:1000); GFP-trap beads are from Chromotek (GFP-Trap A, cat # gta-20); anti-FLAG monoclonal antibody (clone M2) is from Sigma (cat # F3165, 1:2000); anti-FLAG resin is from Sigma (M2 anti-FLAG antibody-linked resin); Hsc/Hsp70 antibodies are from Santa Cruz (cat # sc-1059, 1:1000, sc-1060, 1:1000); Aha1 antibody is from Rockland (cat # 600-401-974); p23 antibody is from Assay Designs (ADI-SPA-610, 1:1000); HOP antibody is from Cell Signaling (cat # 4464, 1:1000); FKBP59 antibody is from StressMarq (SMC-139, 1:1000); Cdk4 antibody is from Santa Cruz (sc-601, 1:1000); HA antibody is from Roche diagnostics (rat anti-HA, clone 3F10, 1:1000); anti-c-Myc Agarose Affinity Gel antibody from Sigma (cat # A7470); and penta·His Antibody, BSA-free, is from Qiagen (cat # 34660, 1:5000).

    Techniques: Binding Assay, Lysis, Immunoprecipitation, SDS Page, Western Blot, Transfection