Anti-PDK3 antibody [EPR29108-32]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling PDK3 with ab322408 at 1/1000 (0.489 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining in human cerebrum.
The section was incubated with ab322408 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-PDK3 antibody [EPR29108-32] (AB322408)

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling PDK3 with ab322408 at 1/1000 (0.489 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining in human prostatic hyperplasia.
The section was incubated with ab322408 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

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Immunocytochemistry/ Immunofluorescence - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling PDK3 with ab322408 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in HepG2 cell line (shown in green). 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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling PDK3 with ab322408 at 1/1000 (0.489 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining in mouse cerebrum.
The section was incubated with ab322408 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-PDK3 antibody [EPR29108-32] (AB322408)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) cells labelling PDK3 with ab322408 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ 1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in C6 cell line (shown in green). 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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ 1000 2ug/ml dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling PDK3 with ab322408 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in NIH/3T3 cell line (shown in green). 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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ 1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling PDK3 with ab322408 at 1/1000 (0.489 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining in rat cerebrum.
The section was incubated with ab322408 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-PDK3 antibody [EPR29108-32] (AB322408)

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

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

All lanes:

Western blot - Anti-PDK3 antibody [EPR29108-32] (ab322408) at 1/2000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

293T transfected with siRNA specifically targeting PDK3 whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) 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: 47 kDa,36 kDa

true

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-PDK3 antibody [EPR29108-32] (AB322408)

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

The expression profile observed is consistent with what has been described in the literature : DFO induced PDK3 expression (PMID : 21763680).

The identity of the bands higher than 75 kDa and lower than 37 kDa are unknown.

Lanes 4-8 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lanes 1-3 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

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

Exposure time : Lane 1 : 180 seconds; Lane 2 : 70 seconds; Lanes 3-7 : 15 seconds; Lane 8 : 3 seconds.

All lanes:

Western blot - Anti-PDK3 antibody [EPR29108-32] (ab322408) at 1/1000 dilution

Lane 1:

Human skeletal muscle tissue lysate at 20 µg

Lane 2:

Human lung tissue lysate at 20 µg

Lane 3:

Human testis tissue lysate at 20 µg

Lane 4:

Untreated HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HCT 116 treated with 0.5 mM Deferoxamine mesylate (DFO) for 24 hours whole cell lysate at 20 µg

Lane 6:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 4 - 8:

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: 47 kDa,36 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-PDK3 antibody [EPR29108-32] (AB322408)

Dot blot analysis of PDK3 using ab322408 at 1 : 1000 (0.489 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : Immunogen peptide of PDK3
Lane2 : His-tagged human PDK1 fragment
Lane3 : His-tagged human PDK2 recombinant protein
Lane4 : His-tagged human PDK4 fragment

Exposure time : 180 seconds.

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

This antibody does not cross-react with recombinant human PDK1, PDK2 or PDK4 by dot blot.

In Dot Blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Dot Blot - Anti-PDK3 antibody [EPR29108-32] (ab322408) at 1/1000 dilution

Lane 1:

Immunogen peptide of PDK3

Lane 2:

His-tagged human PDK1 fragment

Lane 3:

His-tagged human PDK2 recombinant protein

Lane 4:

His-tagged human PDK4 fragment

Secondary

All lanes:

Dot 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

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PDK3 antibody [EPR29108-32] (AB322408)

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

The identity of the bands higher than 75 kDa and lower than 37 kDa are unknown.

All lanes:

Western blot - Anti-PDK3 antibody [EPR29108-32] (ab322408) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

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

Lane 3:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

C6 (rat glial tumor glial cell) 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: 47 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-PDK3 antibody [EPR29108-32] (AB322408)

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

The identity of the bands higher than 75 kDa and lower than 37 kDa are unknown.

All lanes:

Western blot - Anti-PDK3 antibody [EPR29108-32] (ab322408) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Rat testis tissue lysate at 20 µg

Lane 4:

Rat spleen tissue 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: 47 kDa

false

Exposure time: 10s