Anti-SNX27 antibody [1C6]

5 product images

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  Flow Cytometry (Intracellular) - Anti-SNX27 antibody [1C6] (ab77799)

  Overlay histogram showing Jurkat cells stained with ab77799 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab77799, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
  
  

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  Immunoprecipitation - Anti-SNX27 antibody [1C6] (ab77799)

  SNX27 was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Mouse monoclonal to SNX27 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, Hek293 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 ab77799.
  Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
  Band: 61kDa: SNX27.

  All lanes: Immunoprecipitation - Anti-SNX27 antibody [1C6] (ab77799)

  Predicted band size: 61 kDa

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  Western blot - Anti-SNX27 antibody [1C6] (ab77799)

  Blocking: 5% Milk in TBS-T.
  

  Two bands are observed very close together, at around 60-kDa. There are four reported isoforms of the SNX27 protein (Swissprot). We hypothesise that this antibody detects both isoform 1 and isoform 2, which are 61-kDa and 60-kDa respectively. As the immunogen is derived from the N-terminal region of SNX27, the antibody is not expected to react with isoforms 3 or 4.

  All lanes: Western blot - Anti-SNX27 antibody [1C6] (ab77799) at 1 µg/mL

  Lane 1: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 2: A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 3: HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

  Lane 4: A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate at 10 µg

  Lane 5: MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

  Secondary

  All lanes: Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 61 kDa

  Observed band size: 61 kDa

  Exposure time: 15min

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  This image is courtesy of an anonymous customer review

  Western blot - Anti-SNX27 antibody [1C6] (ab77799)

  4-12% bis-tris gel, MES buffer

  All lanes: Western blot - Anti-SNX27 antibody [1C6] (ab77799) at 1/1000 dilution

  All lanes: human kidney whole cell lysate at 30 µg

  Secondary

  All lanes: IRDye® 680RD Goat anti-Mouse IgG (H + L) at 1/15000 dilution

  Performed under reducing conditions.

  Exposure time: 5min

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  Immunocytochemistry/ Immunofluorescence - Anti-SNX27 antibody [1C6] (ab77799)

  ab77799 staining SNX27 in Jurkat cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab77799 at 5µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.