Anti-PDHA1 antibody [EPR11098]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling PDHA1 with unpurified ab168379 at 1/100 dilution. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunocytochemistry/ Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling PDHA1 with Purified ab168379 at 1 : 100 dilution (3.5 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunofluorescent analysis of HepG2 cells labeling PDHA1 with unpurified ab168379 at 1/100 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling PDHA1 with purified ab168379 at 1/40 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling PDHA1 with unpurified ab168379 at 1/100 dilution. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunocytochemistry/Immunofluorescence analysis Jurkat (human acute T cell leukemia) labelling PDHA1 with purified ab168379 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control : PBS only

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling PDHA1 with Purified ab168379 at 1 : 200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Intracellular flow cytometric analysis of permeabilized Jurkat cells labeling PDHA1 (red) with unpurified ab168379 at 1/10 dilution, or a rabbit IgG (negative) (green).

• IP

Lab

Immunoprecipitation - Anti-PDHA1 antibody [EPR11098] (AB168379)

ab168379 (purified) at 1 : 20 dilution (2ug) immunoprecipitating PDHA1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab168379 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab168379 in HeLa (Human cervix adenocarcinoma epithelial cell) 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.

All lanes:

Immunoprecipitation - Anti-PDHA1 antibody [EPR11098] (ab168379)

Predicted band size: 43 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-PDHA1 antibody [EPR11098] (AB168379)

Detection of PDHA1 by Western Blot of Immunprecipitate. 293T cell lysate immunoprecipitated using unpurified ab168379 at 1/10 dilution; HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.

All lanes:

Immunoprecipitation - Anti-PDHA1 antibody [EPR11098] (ab168379)

Predicted band size: 43 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling PDHA1 with Purified ab168379 at 1 : 200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 antibody [EPR11098] (AB168379)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling PDHA1 with Purified ab168379 at 1 : 200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using 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.

• WB

Supplier Data

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

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

In Western blot, Anti-PDHA1 antibody [EPR11098] - (ab168379) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-PDHA1 (phospho S232) antibody [EPR29825-102] (<a href='/en-us/products/primary-antibodies/pdha1-phospho-s232-antibody-epr29825-102-ab325169'>ab325169</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (Lambda Protein Phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

Lanes 1 - 3 : Merged signal (red and green). Green - ab168379 observed at 43 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab168379 was shown to recognize PDHA1 in wild-type A549 cells as signal was lost at the expected MW in PDHA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PDHA1 knockout samples were subjected to SDS-PAGE. ab168379 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/1000 dilution

Lane 1:

Wild-type A549 whole cell lysate at 20 µg

Lane 2:

PDHA1 knockout A549 whole cell lysate at 20 µg

Lane 2:

Western blot - Human PDHA1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-pdha1-knockout-a549-cell-line-ab261866'>ab261866</a>)

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

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

The identity of the band higher than 250 kDa is unknown.

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

In Western blot, Anti-PDHA1 antibody (ab168379) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (<a href='/en-us/products/primary-antibodies/pdha1-phospho-s300-antibody-epr29824-87-ab324265'>ab324265</a>) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

HeLa treated with 100ng/ml Calyculin A for 30 min whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 4:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 6:

HeLa treated with 100ng/ml Calyculin A for 30 min whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 8:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 43 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

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

Performed under reducing conditions.

In Western blot, ab325169 was shown to bind specifically to PDHA1. Target of interest was observed at 43 kDa in induced wild-type A549 cell lysates (lane 2) with no signal observed at this size in induced PDHA1 knockout cell line (lane 4).

Lanes 1-4 on the left untreated membrane with Lambda Protein Phosphatase; Lanes 1-4 on the right treated membrane with Lambda Protein Phosphatase.

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

In Western blot, Anti-PDHA1 antibody [EPR11098] - (ab168379) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-PDHA1 (phospho S232) antibody [EPR29825-102] (<a href='/en-us/products/primary-antibodies/pdha1-phospho-s232-antibody-epr29825-102-ab325169'>ab325169</a>) at 1/1000 dilution

Lane 1:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

A549 treated with 100ng/ml Calyculin A for 30 minutes whole cell lysate at 20 µg

Lane 3:

Untreated PDHA1 knockout A549 whole cell lysate at 20 µg

Lane 4:

PDHA1 knockout A549 treated with 100ng/ml Calyculin A for 30 minutes whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/2000 dilution

Lane 1:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

Mouse brain lysates at 20 µg

Lane 3:

Rat brain lysates at 20 µg

Lane 4:

Mouse kidney lysates at 20 µg

Lane 5:

Rat kidney lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

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

Performed under reducing conditions.

In Western blot, ab324265 was shown to bind specifically toPDHA1 (phospho S300). Target of interest was observed at 43 kDa in Wild-type A549 treated with 100ng/ml Calyculin A (lane 2), with no signal observed at this size in PDHA1 knockout cell line (lanes 3-4).

The identity of the band higher than 250 kDa is unknown.

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

In Western blot, Anti-PDHA1 antibody (ab168379) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (<a href='/en-us/products/primary-antibodies/pdha1-phospho-s300-antibody-epr29824-87-ab324265'>ab324265</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Wild-type A549 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated PDHA1 knockout A549 whole cell lysate (untreated membrane) at 20 µg

Lane 4:

PDHA1 knockout A549 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 6:

Wild-type A549 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated PDHA1 knockout A549 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 8:

PDHA1 knockout A549 treated with 100ng/ml Calyculin A for 30 min whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 43 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

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

In Western blot, Anti-PDHA1 antibody [EPR11098] - (ab168379) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-PDHA1 (phospho S232) antibody [EPR29825-102] (<a href='/en-us/products/primary-antibodies/pdha1-phospho-s232-antibody-epr29825-102-ab325169'>ab325169</a>) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 minutes whole cell lysate (untreated membrane) at 20 µg

Lane 3:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 minutes whole cell lysate (Lambda Protein Phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43 kDa

false

Exposure time: 10s

• WB

Unknown

Western blot - Anti-PDHA1 antibody [EPR11098] (AB168379)

All lanes:

Western blot - Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/1000 dilution

Lane 1:

Human fetal kidney lysate at 10 µg

Lane 2:

A549 lysate at 10 µg

Lane 3:

Jurkat lysate at 10 µg

Lane 4:

HepG2 lysate at 10 µg

Lane 5:

HeLa lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 43 kDa

false