All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/2000 dilution (purified)

Lane 1 : Rat kidney lysates
Lane 2 : Rat kidney lysates.Then the membrane was incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 40 kDa



Blocking and diluting buffer : 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat colon tissue sections labeling PDHA1 with Purified ab177461 at 1:250 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

ab177461 (purified) at 1:20 dilution (2µg) immunoprecipitating PDHA1 in HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate.
Lane 1 (input): HEK-293T whole cell lysate 10µg
Lane 2 (+): ab177461 & HEK-293T whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab177461 in HEK-293T whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent(HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue sections labeling PDHA1 with Purified ab177461 at 1:250 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labeling PDHA1 with Purified ab177461 at 1:250 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/2000 dilution (purified)

Lane 1 : Mouse kidney lysates
Lane 2 : Mouse kidney lysates.Then the membrane was incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 40 kDa



Blocking and diluting buffer : 5% NFDM/TBST

All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/2000 dilution (purified)

Lane 1 : HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 8 mM Sodium butyrate for 24 hours whole cell lysates
Lane 3 : HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 8 mM Sodium butyrate for 24 hours whole cell lysates.Then the membrane was incubated with phosphatase

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 40 kDa



Blocking and diluting buffer : 5% NFDM/TBST

Serially diluted unpurified ab177461 was bound to immobilised human phospho peptide (S293) - or Control peptide (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/1000 dilution (unpurified)

All lanes : HeLa Whole Cell Lysate + Calyculin A (30nM for 20min)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : goat anti-rabbit (green) and goat anti-mouse at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 40 kDa

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour and then treated with either buffer (lane 1) or alkaline phosphatase (lane 2), before being incubated with ab177461 overnight at 4°C. Antibody binding was detected using IR-labelled goat anti-rabbit (green) and goat anti-mouse (Red) at 1:10,000 dilution for one hour at room temperature before imaging.

 

All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/1000 dilution (unpurified)

Lane 1 : Immunoprecipitation pellet from HEK-293T cell lysate at 10 µg
Lane 2 : 1X PBS (negative control)

Secondary
All lanes : Goat-anti-rabbit HRP at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 40 kDa

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling PDHA1 with unpurified  ab177461 at 1/50 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling PDHA1 with unpurified  ab177461 at 1/50 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-PDHA1 (phospho S293) antibody [EPR12200] (ab177461) at 1/1000 dilution (unpurified)

Lane 1 : HEK-293T cell lysates untreated
Lane 2 : HEK-293T cell lysates, membrane treated with Lambda Phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat-anti-rabbit HRP at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 40 kDa