Anti-PDHA1 (phospho S300) antibody [EPR29824-87]

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human colon tissue staining PDHA1 (phospho S300) with ab324265 at a 1 : 500 (0.994 ug/ml) dilution, ab202554 anti-COX4 used at 1 : 500 (0.228 ug/ml) dilution.

Panel A : merged staining of anti-PDHA1 (phospho S300) (green; Opal™520), anti-COX4 (magenta; Opal™570) on human colon.
Panel B : anti-PDHA1 (phospho S300) showed granular staining in human colon.
Panel C : anti-COX4 showed granular staining in human colon.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab324265, ab202554 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Human colon without alkaline phosphatase treatment (B) Human colon with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) human colon without alkaline phosphatase treatment; negative staining on (B) human colon with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Human endometrial carcinoma without alkaline phosphatase treatment (B) Human endometrial carcinoma with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) human endometrial carcinoma without alkaline phosphatase treatment; negative staining on (B) human endometrial carcinoma with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PDHA1 KO A549 (PDHA1 knockout human lung carcinoma epithelial cell)(ab261866) cells labelling PDHA1 (phospho S300) with ab324265 at 1/200 (2.485 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in A549 cell line (shown in green) treated with Calyculin A (100 ng/mL) for 30 min, and no staining after alkaline phosphatase (AP) treatment.
A : parental A549 cells
B : parental A549 cells treated with Calyculin A (100 ng/mL) for 30 min
C : parental A549 cells treated with Calyculin A (100 ng/mL) for 30 min after alkaline phosphatase (AP) treatment
D : PDHA1 knockout A549 cells treated with Calyculin A (100 ng/mL) for 30 min
The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/ab7291 1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Human endometrium without alkaline phosphatase treatment (B) Human endometrium with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) human endometrium without alkaline phosphatase treatment; negative staining on (B) human endometrium with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Human astrocytoma without alkaline phosphatase treatment (B) Human astrocytoma with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) human astrocytoma without alkaline phosphatase treatment; negative staining on (B) human astrocytoma with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Mouse colon without alkaline phosphatase treatment (B) Mouse colon with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse colon without alkaline phosphatase treatment; negative staining on (B) mouse colon with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Mouse spleen without alkaline phosphatase treatment (B) Mouse spleen with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse spleen without alkaline phosphatase treatment; negative staining on (B) mouse spleen with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Mouse lung papillary adenocarcinoma without alkaline phosphatase treatment (B) Mouse lung papillary adenocarcinoma with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse lung papillary adenocarcinoma without alkaline phosphatase treatment; negative staining on (B) mouse lung papillary adenocarcinoma with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Immunohistochemical analysis of paraffin-embedded (A) Mouse hepatocellular carcinoma without alkaline phosphatase treatment (B) Mouse hepatocellular carcinoma with alkaline phosphatase treatment tissue labeling PDHA1 (phospho S300) with ab324265 at 1/500 (0.994 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse hepatocellular carcinoma without alkaline phosphatase treatment; negative staining on (B) mouse hepatocellular carcinoma with alkaline phosphatase treatment. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Supplier Data

Western blot - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

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] (ab324265) 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='/ko/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 (phospho S300) antibody [EPR29824-87] (AB324265)

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] (ab324265) 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='/ko/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

• Dot

Supplier Data

Dot Blot - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (AB324265)

Dot blot analysis of PDHA1 (phospho S300) using ab324265 at 1000 (0.497 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : PDHA1 (phospho S300) peptide a
Lane2 : PDHA1 (phospho S300) peptide b
Lane3 : PDHA1 non-phospho peptide c
Lane4 : PDHA1 (phospho Y301) peptide d
Lane5 : PDHA1 (phospho T303) peptide e
Lane6 : PDHA1 (phospho S295) peptide f

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-PDHA1 (phospho S300) antibody [EPR29824-87] (ab324265) at 1/1000 dilution

Lane 1:

PDHA1 (phospho S300) peptide a

Lane 2:

PDHA1 (phospho S300) peptide b

Lane 3:

PDHA1 non-phospho peptide c

Lane 4:

PDHA1 (phospho Y301) peptide d

Lane 5:

PDHA1 (phospho T303) peptide e

Lane 6:

PDHA1 (phospho S295) peptide f

Secondary

All lanes:

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

false

Exposure time: 180s