Anti-RBCK1 antibody [EPR28157-53]

7 product images

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  Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Western blot: Anti-RBCK1 antibody [EPR28157-53] (ab309104) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab309104 was shown to bind specifically to RBCK1. A band was observed at 58 kDa in wild-type A549 cell lysates with no signal observed at this size in RBCK1 knockout cell line. To generate this image, wild-type and RBCK1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: RBCK1 knockout A549 cell lysate at 20 µg

  Lane 3: HepG2 cell lysate at 20 µg

  Lane 4: MCF7 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 58 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling RBCK1 with ab309104 at 1/50 (10.14 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing mostly cytoplasmic staining in HepG2 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

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  Flow Cytometry (Intracellular) - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling RBCK1 with ab309104 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.

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  Flow Cytometry (Intracellular) - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling RBCK1 with ab309104 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.

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  Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  The expression molecular weight observed is consistent with what has been described in the literature (PMID: 31209050, PMID: 27006117).
  The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID: 31209050).
  Bands below 57kd are cleaved RBCK1(PMID: 27006117).
  The blot of lanes1, 2 and 6-8 were developed using a high sensitivity ECL substrate.
  Exposure time: Lanes1, 2 and 6-8: 180 seconds; Lane3-5: 48 seconds

  All lanes: Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104) at 1/1000 dilution

  Lane 1: Human hypothalamus tissue lysate at 20 µg

  Lane 2: Human spinal cord tissue lysate at 20 µg

  Lane 3: Mouse brain tissue lysate at 20 µg

  Lane 4: Mouse spleen tissue lysate at 20 µg

  Lane 5: Mouse kidney tissue lysate at 20 µg

  Lane 6: Rat brain tissue lysate at 20 µg

  Lane 7: Rat spleen tissue lysate at 20 µg

  Lane 8: Rat kidney tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 57 kDa

• 

  Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  The expression molecular weight observed is consistent with what has been described in the literature (PMID: 31209050, PMID: 27006117).
  The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID: 31209050).
  Bands below 57kd are cleaved RBCK1(PMID: 27006117).
  In Western blot, anti- H3 antibody (Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade ab176842) loading control staining at 1/100000 dilution.

  All lanes: Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104) at 1/1000 dilution

  Lane 1: HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: HepG2 transfected with siRNA specifically targeti RBCK1 whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 57 kDa

  Exposure time: 59s

• 

  Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  The expression molecular weight observed is consistent with what has been described in the literature (PMID: 31209050, PMID: 27006117).
  The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID: 31209050).
  Bands below 57kd are cleaved RBCK1(PMID: 27006117).

  All lanes: Western blot - Anti-RBCK1 antibody [EPR28157-53] (ab309104) at 1/1000 dilution

  Lane 1: A549 (human lu carcinoma epithelial cell)whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

  Lane 3: C2C12 (mouse myoblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 57 kDa

  Exposure time: 180s