Anti-PFKP antibody [EPR28830-157]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PFKP antibody [EPR28830-157] (AB325016)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Parental HeLa (EDWT03) (Green) / PFKP KO HeLa (ED030126) (Magenta) cells labelling PFKP with ab325016 at 1/500 dilution (0.1ug) (Magenta) and Green compared with a Rabbit monoclonal IgG (ab172730) (Black) and Grey isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PFKP antibody [EPR28830-157] (AB325016)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PFKP KO HeLa (PFKP knockout human cervical adenocarcinoma epithelial cell))(ab265136) cells labelling PFKP with ab325016 at 1/200 (2.565 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 and weak nuclear staining in parental HeLa 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).

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).

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

• IP

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Immunoprecipitation - Anti-PFKP antibody [EPR28830-157] (AB325016)

PFKP was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab325016 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325016 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-PFKP antibody [EPR28830-157] (ab325016) at 1/30 dilution

Lanes 1 - 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325016 in HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 85 kDa

false

Exposure time: 41s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PFKP antibody [EPR28830-157] (AB325016)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized P815 (mouse mastocytoma mast cell) cells labelling PFKP with ab325016 at 1/50 (10.26 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 P815 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).

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).

-ve control 1 : ab325016 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• WB

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Western blot - Anti-PFKP antibody [EPR28830-157] (AB325016)

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-PFKP antibody [EPR28830-157] (ab325016) at 1/1000 dilution

Lane 1:

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

Lane 2:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysat at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

DU 145 (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 6:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 85 kDa,36 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-PFKP antibody [EPR28830-157] (AB325016)

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

Low expression : liver

Lanes 1-2 are incubated with are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lanes 3-11 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000.

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

All lanes:

Western blot - Anti-PFKP antibody [EPR28830-157] (ab325016) at 1/1000 dilution

Lane 1:

Human colon cancer tissue lysate at 20 µg

Lane 2:

Human lung cancer tissue lysate at 20 µg

Lane 3:

Mouse testis tissue lysate at 20 µg

Lane 4:

Rat testis tissue lysate at 20 µg

Lane 5:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 6:

P815 (mouse mastocytoma mast Cell ) whole cell lysate at 20 µg

Lane 7:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 8:

Mouse brain tissue lysate at 20 µg

Lane 9:

Mouse liver tissue lysate at 20 µg

Lane 10:

Rat brain tissue lysate at 20 µg

Lane 11:

Rat liver tissue lysate at 20 µg

Secondary

Lanes 1 - 2:

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

Lanes 3 - 11:

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

Observed band size: 85 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PFKP antibody [EPR28830-157] (AB325016)

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

Performed under reducing conditions.

In Western blot, ab325016 was shown to bind specifically to PFKP. Target of interest was observed at 85 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in PFKP knockout cell line (lane 2) (lane 2, knockout cell line ab265136/ knockout cell lysate ab257580).

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

All lanes:

Western blot - Anti-PFKP antibody [EPR28830-157] (ab325016) at 1/1000 dilution

Lane 1:

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

Lane 2:

PFKP knockout HeLa whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 85 kDa,36 kDa

false

Exposure time: 180s

• I-ELISA

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Indirect ELISA - Anti-PFKP antibody [EPR28830-157] (AB325016)

Indirect ELISA analysis of ab325016 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human PFKP peptide, Human PFKM peptide, Human PFKL peptide.

Antigen concentration : 1000 ng/ml