Anti-P4HB antibody [EPR9499]

17 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [EPR9499] (ab137110)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Western blot - Anti-P4HB antibody [EPR9499] (ab137110)

  False colour image of Western blot: Anti-P4HB antibody [EPR9499] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab137110 was shown to bind specifically to P4HB. A band was observed at 60 kDa in wild-type HeLa cell lysates with no signal observed at this size in P4HB knockout cell line Human P4HB knockout A-431 cell line ab261887 (knockout cell lysate Human P4HB knockout A-431 cell lysate ab261696). To generate this image, wild-type and P4HB knockout HeLa 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 (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-P4HB antibody [EPR9499] (ab137110) at 1/1000 dilution

  Lane 1: HeLa cell lysate at 20 µg

  Lane 2: HepG2 cell lysate at 20 µg

  Lane 3: Wild-type A431 cell lysate at 20 µg

  Lane 4: P4HB knockout A431 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 57 kDa

  Observed band size: 60 kDa

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  Western blot - Anti-P4HB antibody [EPR9499] (ab137110)

  Blocking and dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution

  Lane 1: HepG2 whole cell lysate at 10 µg

  Lane 2: HeLa whole cell lysate at 10 µg

  Lane 3: HEK293 whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 57 kDa

  Observed band size: 57 kDa

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  Western blot - Anti-P4HB antibody [EPR9499] (ab137110)

  Blocking and dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution

  Lane 1: Raw264.7 whole cell lysate at 10 µg

  Lane 2: PC-12 whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 57 kDa

  Observed band size: 57 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [EPR9499] (ab137110)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [EPR9499] (ab137110)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Flow Cytometry (Intracellular) - Anti-P4HB antibody [EPR9499] (ab137110)

  Intracellular Flow Cytometry analysis ofHeLa cells labelling P4HB with purified ab137110 at a dilution of 1/300 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^®488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

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  Western blot - Anti-P4HB antibody [EPR9499] (ab137110)

  All lanes: Western blot - Anti-P4HB antibody [EPR9499] (ab137110) at 1/1000 dilution

  Lane 1: HepG2 cell lysate at 10 µg

  Lane 2: HeLa cell lysate at 10 µg

  Lane 3: 293T cell lysate at 10 µg

  Lane 4: A431 cell lysate at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

  Developed using the ECL technique.

  Predicted band size: 57 kDa

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  Western blot - Anti-P4HB antibody [EPR9499] (ab137110)

  All lanes: Western blot - Anti-P4HB antibody [EPR9499] (ab137110) at 1/2000 dilution

  Lane 1: Mouse brain tissue lysate

  Lane 2: Mouse heart tissue lysate

  Lane 3: Mouse kidney tissue lysate

  Lane 4: Mouse spleen tissue lysate

  Predicted band size: 57 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [EPR9499] (ab137110)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.
  

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [EPR9499] (ab137110)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.
  

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-P4HB antibody [EPR9499] (ab137110)

  Overlay histogram showing HepG2 cells stained with unpurified ab137110 (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 (unpurified ab137110, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor^® 488 (IgG H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HepG2 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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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [EPR9499] (ab137110)

  ab137110 staining P4HB in A549 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab137110 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [EPR9499] (ab137110)

  ab137110 staining P4HB in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab137110 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [EPR9499] (ab137110)

  ab137110 staining P4HB in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab137110 at 1µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [EPR9499] (ab137110)

  ab137110 staining P4HB in U2OS cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab137110 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [EPR9499] (ab137110)

  ab137110 staining P4HB in wild-type A431 cells (top panel) and P4HB knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab137110 at 1µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.