重组Anti-PFKFB3抗体[EPR12594] (ab181861)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: PFKFB3 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Jurkat whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab181861 observed at 60 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab181861 was shown to specifically react with PFKFB3 in wild-type HAP1 cells as signal was lost in PFKFB3 knockout cells. Wild-type and PFKFB3 knockout samples were subjected to SDS-PAGE. Ab181861 and ab9484 (Mouse anti GAPDH 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.

Immunofluorescent analysis of acetone-fixed HeLa cells labeling PFKFB3 with ab181861 at 1/100 dilution, followed by Goat anti rabbit IgG (Alexa Fluor®555) at 1/200 dilution. Counter stained with Dapi (blue).

Blocking buffer:  5% NFDM/TBST

Blocking buffer:  5% NFDM/TBST

Exposure time: 15 seconds

This blot was developed using a high sensitivity ECL substrate.

Exposure time: Lane 1-2: 26 seconds, Lane 3-7: 48 seconds

Blocking buffer and concentration: 5% NFDM/TBST

Lane 3-7 were developed using a high sensitivity ECL substrate.
The expression level of PFKFB3 is upregulated during 3T3-L1 differentiation (PMID: 16306349).
The band at approximately 110 kDa is likely to be PFKFB3 dimer (PMID: 31889092).

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling PFKFB3 with ab181861 at 1/50 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling PFKFB3 with purified ab181861 at 1/210 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control. 

Western blot analysis of PFKFB3 in HeLa cell lysate immunoprecipitated using ab181861 at 1/50 dilution (Lane 1). Lane 2: Negative control.

Secondary antibody: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500 dilution.

PFKFB3 was immunoprecipitated from 0.35 mg of Mouse skin tissue lysate with ab181861 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab181861 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: Mouse skin tissue lysate 10 μg (Input). 

Lane 2: ab181861 IP in Mouse skin tissue lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab181861 in Mouse skin tissue lysate

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

Exposure time: 180 seconds.

This blot was developed using a high sensitivity ECL substrate.

PFKFB3 was immunoprecipitated from 0.35 mg of AR42J (rat pancreatic tumor epithelial cell) whole cell lysate with ab181861 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab181861 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: AR42J (rat pancreatic tumor epithelial cell) whole cell lysate 10 μg (Input). 

Lane 2: ab181861 IP in AR42J whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab181861 in AR42J whole cell lysate

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

Exposure time: 180 seconds.

Immunofluorescent analysis of 4% paraformaldehyde-fixed A431 cells labeling PFKFB3 with ab181861 at 1/100 dilution, followed by Goat anti rabbit IgG (Alexa Fluor®555) at 1/200 dilution. Counter stained with Dapi (blue).