Anti-ZW10 antibody

5 product images

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  Immunoprecipitation - Anti-ZW10 antibody (ab21582)

  ZW10 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to ZW10 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab21582.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).Band: 88kDa: ZW10

  All lanes: Immunoprecipitation - Anti-ZW10 antibody (ab21582)

  Predicted band size: 88 kDa

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  This image is courtesy of Scott Slattery and Mike Mancini

  Immunocytochemistry/ Immunofluorescence - Anti-ZW10 antibody (ab21582)

  Panel one shows staining of HeLa cells by anti-ZW10 (ab21582) in green and DAPI in blue. The second panel shows staining by anti-ZW10 in green and SH-CREST in red, which stains the centromeres. In most cells at interphase, ZW10 is present diffusely in the cytoplasm. In prometaphase, it is associated with the kinetochore. Fix cells for 30 minutes on ice in 4% formaldehyde in PEM.
  Quench autofluorescence 2 x 5 minutes with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM.
  Permeablize 30 minutes with 0.5% TX-100 in PEM.
  Block 30 minutes in 5% milk in TBST.
  Primary antibody incubated overnight at 4oC diluted 1/100 in 5% milk in TBST.
  Secondary antibody 1 hour at RT diluted in 5% milk in TBST.
  Post-fix 20 min. on ice in 4% formaldehyde in PEM.
  Quench autofluorescence 2 x 5 minutes with ammonium chloride in PEM.
  Counterstain with DAPI in TBST.
  Mount with ProLong Gold antifade reagent from Invitrogen.
  Notes: Ample washing between each step. TBST = Tris buffered saline +

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  Western blot - Anti-ZW10 antibody (ab21582)

  This antibody detects a band at approximately 88kDa that corresponds in size to ZW10. It also detects bands at 75 and 45kDa that may be degradation products or crossreactivity. All of these bands are competed away by the addition of the immunizing peptide, suggesting that they are specific interactions.

  All lanes: Western blot - Anti-ZW10 antibody (ab21582) at 1 µg/mL

  All lanes: Western blot - Jurkat whole cell lysate (Jurkat whole cell lysate ab7899) at 20 µg

  Secondary

  All lanes: Goat polyclonal to Rabbit IgG(Alexa Fluor® 680) at 1/10,000 dilution

  Performed under reducing conditions.

  Predicted band size: 88 kDa

  Observed band size: 45 kDa, 75 kDa, 88 kDa

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  This image is courtesy of a customer review submitted by Dr Kirk McManus

  Immunocytochemistry/ Immunofluorescence - Anti-ZW10 antibody (ab21582)

  ab21582 (1/200) staining ZW10 in asynchronous mouse 10T1/2 cells (green). Cells were pre-extracted with PEM (1min) at room temperature, fixed in paraformaldehyde (10 mins), washed in KB+ buffer and counterstained with DAPI in order to highlight the DNA/nucleus (red). Please note that ab21582 might only perform well when using this specific protocol. Please refer to abreview for further experimental details.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-ZW10 antibody (ab21582)

  ZW10 western blot using anti-ZW10 antibody ab21582. Publication image and figure legend from Otterpohl, K. L. & Gould, K. A., 2017, PLoS One, PubMed 28264000.

  ab21582 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab21582 please see the product overview.

  In vitro characterization of missense variants in RINT1.FLAG-tagged RINT1 mutant plasmids were transfected into HEK293T cells and an anti-FLAG antibody was then used to immunoprecipitate RINT1 and interacting proteins from cell lysate. Cells transfected with empty vector served as the negative control (Neg Con). Cells transfected with the RINT1 wildtype (WT) expression vector served as the positive control. A representative immunoblot for ZW10 immunoprecipitation is shown (A). Quantification of the ZW10 immunoprecipitation blots (N = 5) are shown (B). A representative immunoblot for UVRAG immunoprecipitation is shown (C). Quantification of the UVRAG immunoprecipitation blots (N = 4) are shown (B). The asterisk indicates differences with a p ≤ 0.05.