Anti-P4HB antibody [RL90]

19 product images

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  Flow Cytometry - Anti-P4HB antibody [RL90] (ab2792)

  Overlay histogram showing HeLa cells stained with ab2792 (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 (ab2792, 0.5μg/1x10⁶ cells) for 30 min at 22°C. 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 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (Mouse IgG2a, Kappa Monoclonal [B12/8] - Isotype Control ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  
  

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  Western blot - Anti-P4HB antibody [RL90] (ab2792)

  All lanes: Western blot - Anti-P4HB antibody [RL90] (ab2792) at 1/2000 dilution

  Lane 1: HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 30 µg

  Lane 2: A431 (human epidermoid carcinoma cell line) whole cell lysate at 30 µg

  Lane 3: NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 30 µg

  Lane 4: A-375 (human malignant melanoma cell line) whole cell lysate at 30 µg

  Lane 5: HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 30 µg

  Lane 6: HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 30 µg

  Lane 7: PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 30 µg

  Secondary

  All lanes: Goat anti-Mouse IgG H+L (HRP) at 1/4000 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 [RL90] (ab2792)

  Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human colon tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing P4HB (PDIA1) ab2792 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
  
  

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  Western blot - Anti-P4HB antibody [RL90] (ab2792)

  Western blot analysis of P4HB (PDIA1) was performed by loading 25 ug of HepG2 (Lane 1) Hela (Lane 2) and NIH-3T3 (Lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with ab2792 at 1:1000 overnight at 4°C and washed in TBST. The membrane was then probed with a HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using a ECL Plus Western Blotting Substrate. Results show a band at approx. 57 kDa.

  All lanes: Western blot - Anti-P4HB antibody [RL90] (ab2792)

  Predicted band size: 57 kDa

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  This image is courtesy of an anonymous customer review

  Western blot - Anti-P4HB antibody [RL90] (ab2792)

  All lanes: Western blot - Anti-P4HB antibody [RL90] (ab2792) at 1/2000 dilution

  Secondary

  All lanes: Donkey anti mouse IgG2a at 1/10000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 57 kDa

  Observed band size: 57 kDa

  Exposure time: 20s

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  Western blot - Anti-P4HB antibody [RL90] (ab2792)

  10% SDS-PAGE.
  

  Blocked with 5% milk for 1 hour at 22°C.

  Incubated with the primary for 16 hours at 4°C in PBS + 2.5% milk + 0.05% Tween20.

  All lanes: Western blot - Anti-P4HB antibody [RL90] (ab2792) at 1/1000 dilution

  All lanes: HT1080 whole cell lysate at 20000 Cells

  Secondary

  All lanes: HRP-conjugated sheep anti-mouse IgG at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 57 kDa

  Observed band size: 57 kDa

  Exposure time: 20s

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  Flow Cytometry - Anti-P4HB antibody [RL90] (ab2792)

  Flow cytometry analysis of P4HB (PDIA1) showing positive staining in the membrane and cytoplasm of K562 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2792 at 1 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

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  This image is courtesy of an anonymous customer review

  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [RL90] (ab2792)

  ab2792 staining P4HB (PDIA1) in MDA MB 231 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 1% Triton X-100 and blocked with 10% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/100 in BSA + 0.02% Tween 20) for 1 hour at 16°C. A DyLight^® 550-conjugated goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody.

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  Flow Cytometry - Anti-P4HB antibody [RL90] (ab2792)

  Flow cytometry analysis of P4HB (PDIA1) showing positive staining in the membrane and cytoplasm of NIH/3T3 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2792 at 0.5 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

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  Flow Cytometry - Anti-P4HB antibody [RL90] (ab2792)

  Flow cytometry analysis of P4HB (PDIA1) showing positive staining in the membrane and cytoplasm of Hela cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2792 at 1 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [RL90] (ab2792)

  Immunocytochemistry/Immunofluorescence analysis of P4HB (PDIA1) (green) in NIH 3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with ab2792 (1:75) for at least 1 hour at room temperature and incubated with Dylight 488 goat anti-mouse IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin was stained with Dylight 350 Phalloidin at a dilution of 1:120 (2.5units/ml final concentration) and nuclei (red) were stained with DRAQ5 at a concentration of 1ug/ml for 30 minutes. Images were taken at 20X magnification.

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  This image is a courtesy of Anonymous customer review

  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [RL90] (ab2792)

  ab2792 staining P4HB (PDIA1) from human HaCaT keratinocyte cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.25 % Triton ×100 and blocking with 2.5% BSA plus 1% goat serum for 1 hour at 4°C was performed. Samples were incubated with primary antibody, diluted 1/100, for 1 hour at 240C. An Alexa Fluor^® 488-conjugated goat polyclonal to mouse IgG was used at dilution at 1/1000 as secondary antibody.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [RL90] (ab2792)

  Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human tonsil tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing P4HB (PDIA1) ab2792 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
  
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-P4HB antibody [RL90] (ab2792)

  Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human lung adenocarcinoma tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing P4HB (PDIA1) ab2792 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
  
  

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  Immunocytochemistry/ Immunofluorescence - Anti-P4HB antibody [RL90] (ab2792)

  ab2792 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 ab2792 at 0.2µg/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) preadsorbed ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed 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 [RL90] (ab2792)

  ab2792 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 ab2792 at 1µg/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) preadsorbed ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed 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 [RL90] (ab2792)

  ab2792 staining P4HB in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 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 ab2792 at 0.2µg/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) preadsorbed ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 100% methanol (5 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 [RL90] (ab2792)

  ab2792 staining P4HB in A549 cells. The cells were fixed with 4% paraformaldehyde (10 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 ab2792 at 0.2µg/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) preadsorbed ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 100% methanol (5 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 [RL90] (ab2792)

  ab2792 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 ab2792 at 1µg/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) preadsorbed ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed 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.