Anti-PFKFB3 antibody [EPR12594]

• IP

Lab

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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.

All lanes:

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861)

Lane 1:

AR42J (rat pancreatic tumor epithelial cell) whole cell lysate at 10 µg

Lane 2:

Anti-PFKFB3 antibody IP in AR42J whole cell lysate

Secondary

All lanes:

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

Predicted band size: 60 kDa

Observed band size: 60 kDa

false

• IP

Lab

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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 at 1/30 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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1:

Mouse skin tissue lysate at 10 µg

Lane 2:

ab181861 at 1/30 IP in Mouse skin tissue lysate at 1/30 dilution

Secondary

All lanes:

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

Observed band size: 60 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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).

All lanes:

Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

bEnd.3 (mouse brain endothelial cell), whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

Undifferentiated 3T3-L1 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 6:

3T3-L1 (mouse embryonic fibroblast) differentiated into adipocyte-like cells, whole cell lysate at 20 µg

Lane 7:

C2C12 (mouse myoblast), 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

Predicted band size: 60 kDa

Observed band size: 60 kDa

true

• WB

Lab

Western blot - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Mouse skin tissue lysate at 20 µg

Lane 3:

Rat breast tissue lysate at 20 µg

Lane 4:

AR42J (rat pancreatic tumor epithelial cell) whole cell lysate at 20 µg

Lane 5:

L6 (rat skeletal muscle myoblast) 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

Predicted band size: 60 kDa

Observed band size: 60 kDa

true

Exposure time: 15s

• WB

Lab

Western blot - Anti-PFKFB3 antibody [EPR12594] (AB181861)

Low expression : kidney tissue (PMID : 34660743).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

All lanes:

Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1:

Mouse lung tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Lane 3:

Mouse heart tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Rat lung tissue lysate at 20 µg

Lane 6:

Rat liver tissue lysate at 20 µg

Lane 7:

Rat heart tissue lysate at 20 µg

Lane 8:

Rat kidney 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: 60 kDa

true

Exposure time: 180s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (AB181861)

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.

All lanes:

Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861)

Predicted band size: 60 kDa

false