Anti-RNF5/NG2 antibody [EPR27133-30]

6 product images

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  Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  RNF5/NG2 Western blot staining using rabbit Anti-RNF5/NG2 antibody

  Western blot: Rabbit Monoclonal[EPR27133-30] to RNF5/NG2 ab308066 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 18 kDa in Wild-type A549 cell lysates with no signal observed at this size in RNF5 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: RNF5 knockout A549 at 20 µg

  Lane 3: SH-SY5Y at 20 µg

  Lane 4: A-498 at 20 µg

  Secondary

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

  Performed under reducing conditions.

  Predicted band size: 20 kDa

  Observed band size: 18 kDa

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  Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.

  All lanes: Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

  Lane 1: Human liver tissue lysate at 20 µg

  Lane 2: Human colon tissue lysate at 20 µg

  Lane 3: Human colon cancer 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

  Developed using the ECL technique.

  Observed band size: 19 kDa

  Exposure time: 70s

• 

  Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.

  All lanes: Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

  Lane 1: 293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

  Lane 2: 293T transfected with siRNA specifically targeting RNF5/NG2 whole cell lysate at 20 µg

  Lane 3: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: MCF7 (human breast adenocarcinoma epithelial cell) 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

  Developed using the ECL technique.

  Observed band size: 19 kDa

  Exposure time: 180s

• 

  Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  
  This antibody does not cross-react with human RNF185.
  
  In Western blot, anti-GST antibody (Anti-GST antibody [EPR4236] ab111947) staining at 1/5000 dilution.
  
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

  Lane 1: Human RNF5 recombinant protein

  Lane 2: Human RNF185 recombinant protein

  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: 45 kDa

  Exposure time: 180s

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  Flow Cytometry (Intracellular) - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling RNF5/NG2 with ab308066 at 1/50 dilution (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. Cells were stained with rabbit IgG or ab308066.

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  Immunoprecipitation - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066)

  RNF5/NG2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab308066 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308066 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  
  Lane 1: HeLa whole cell lysate
  
  Lane 2: ab308066 IP in HeLa whole cell lysate
  
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab308066 in HeLa whole cell lysate
  
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: 10 seconds.

  All lanes: Immunoprecipitation - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/30 dilution

  All lanes: HeLa whole cell lysate at 10 µg

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 19 kDa

  Exposure time: 10s