Name: Anti-PFKFB3 antibody [EPR12594]
Brand: RabMAb
SKU: ab181861
Rating: 5 (6 reviews)

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Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR12594] to PFKFB3
Suitable for: Flow Cyt (Intra), WB, IP, ICC/IF, IHC-P
Knockout validated
Reacts with: Mouse, Rat, Human

Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free

Product name

Anti-PFKFB3 antibody [EPR12594]
See all PFKFB3 primary antibodies
Description

Rabbit monoclonal [EPR12594] to PFKFB3
Host species

Rabbit
Tested applications

Suitable for: Flow Cyt (Intra), WB, IP, ICC/IF, IHC-Pmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: Jurkat and HeLa whole cell lysate (ab150035); Human melanoma tissue; HeLa and A431 cells, Mouse skin tissue lysate, Rat breast tissue lysate, AR42 and L6 whole cell lysates, HAP1 whole cell lysate, AR42J (rat pancreatic tumor epithelial cell) whole cell lysate, IP: Mouse skin tissue lysate, AR42J, whole cell lysate ICC: HeLa, A431 cells IHC: human melanoma tissue Flow: A431 (human epidermoid carcinoma) cells,
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR12594
Isotype

IgG
Research areas

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab181861 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
Flow Cyt (Intra)		Use at an assay dependent concentration.
WB	(5)	1/1000 - 1/10000. Detects a band of approximately 58 kDa (predicted molecular weight: 60 kDa).
IP		1/50.
ICC/IF		1/100.
IHC-P		1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Notes
Flow Cyt (Intra) Use at an assay dependent concentration.
WB 1/1000 - 1/10000. Detects a band of approximately 58 kDa (predicted molecular weight: 60 kDa).
IP 1/50.
ICC/IF 1/100.
IHC-P 1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Function

Synthesis and degradation of fructose 2,6-bisphosphate.
Tissue specificity

Ubiquitous.
Sequence similarities

In the C-terminal section; belongs to the phosphoglycerate mutase family.
Post-translational
modifications

Phosphorylation by AMPK stimulates activity.
Target information above from: UniProt accession Q16875 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 5209 Human
- Entrez Gene: 170768 Mouse
- Entrez Gene: 117276 Rat
- Omim: 605319 Human
- SwissProt: Q16875 Human
- SwissProt: A2AUP1 Mouse
- SwissProt: O35552 Rat
- Unigene: 195471 Human
Alternative names
- 6 phosphofructo 2 kinase/ fructose 2,6 bisphosphatase antibody
- 6 phosphofructo 2 kinase/fructose 2,6 biphosphatase 3 antibody
- 6-bisphosphatase antibody
- 6-P2ase 3 antibody
- 6-P2ASE brain/placenta-type isozyme antibody
- 6PF 2 K/Fru 2,6 P2ASE brain/placenta type isozyme antibody
- 6PF 2-K/Fru 2,6 P2ase 3 antibody
- 6PF-2-K/Fru-2 antibody
- F263_HUMAN antibody
- fructose 6 phosphate,2 kinase/fructose 2, 6 bisphosphatase antibody
- Fructose-2 antibody
- Inducible 6 phosphofructo 2 kinase/fructose 2,6 bisphosphatase antibody
- iPFK 2 antibody
- iPFK-2 antibody
- IPFK2 antibody
- PFK/FBPase 3 antibody
- PFK2 antibody
- PFKFB3 antibody
- Renal carcinoma antigen NY REN 56 antibody
- Renal carcinoma antigen NY-REN-56 antibody
- uPFK antibody
see all

Western blot - Anti-PFKFB3 antibody [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.
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).
Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)

All lanes : Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/20000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)

All lanes : Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1 : A431 (human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 2 : Mouse skin tissue lysate
Lane 3 : Rat breast tissue lysate
Lane 4 : AR42J (rat pancreatic tumor epithelial cell) whole cell lysate
Lane 5 : L6 (rat skeletal muscle myoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 60 kDa

Exposure time: 15 seconds

Blocking buffer: 5% NFDM/TBST

Exposure time: 15 seconds

This blot was developed using a high sensitivity ECL substrate.
Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)

All lanes : Anti-PFKFB3 antibody [EPR12594] (ab181861) at 1/1000 dilution

Lane 1 : A431 (human epidermoid carcinoma epithelial cell), whole cell lysate
Lane 2 : bEnd.3 (mouse brain endothelial cell), whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 4 : 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate
Lane 5 : Undifferentiated 3T3-L1 (mouse embryonic fibroblast), whole cell lysate
Lane 6 : 3T3-L1 (mouse embryonic fibroblast) differentiated into adipocyte-like cells, whole cell lysate
Lane 7 : C2C12 (mouse myoblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 60 kDa

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

Flow cytometry protocols
Immunoprecipitation protocols
Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

Publishing research using ab181861? Please let us know so that we can cite the reference in this datasheet.

ab181861 has been referenced in 57 publications.

Fuhrmann DC & Brüne B miR-193a-3p increases glycolysis under hypoxia by facilitating Akt phosphorylation and PFKFB3 activation in human macrophages. Cell Mol Life Sci 79:89 (2022). PubMed: 35072776
He B et al. Long noncoding RNA LINC00930 promotes PFKFB3-mediated tumor glycolysis and cell proliferation in nasopharyngeal carcinoma. J Exp Clin Cancer Res 41:77 (2022). PubMed: 35209949
Wang W et al. Circ_0014130 is involved in the drug sensitivity of colorectal cancer through miR-197-3p/PFKFB3 axis. J Gastroenterol Hepatol 37:908-918 (2022). PubMed: 35288979
Min J et al. β-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress. Commun Biol 5:248 (2022). PubMed: 35318430
Hu J et al. Inhibition of AMPK/PFKFB3 mediated glycolysis synergizes with penfluridol to suppress gallbladder cancer growth. Cell Commun Signal 20:105 (2022). PubMed: 35842652

View all Publications for this product

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Application

IHC - Wholemount

Sample

Mouse Tissue (Lung)

Specification

Lung

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

DR. Ming Jen Hsu

Verified customer

Submitted Aug 18 2021

Application

Western blot

Sample

Mouse Tissue lysate - whole (brain)

Gel Running Conditions

Reduced Denaturing (TEO TRICINE 4-20% gel (ab119209))

Loading amount

20 µg

Specification

brain

Blocking step

BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 25°C

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Dr. Yonatan Feuermann

Verified customer

Submitted Apr 12 2021

Application

Western blot

Sample

Mouse Cell lysate - whole cell (Murine breast cancer cells)

Gel Running Conditions

Reduced Denaturing (10% gel)

Loading amount

5 µg

Specification

Murine breast cancer cells

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 27°C

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Alyssa La Belle

Verified customer

Submitted May 21 2019

Application

Western blot

Sample

Cow Cell lysate - whole cell (VVEC)

Gel Running Conditions

Reduced Denaturing (10)

Loading amount

20 µg

Treatment

100 uM ATP 0, 5 min, 30 min

Specification

VVEC

Blocking step

BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jun 03 2016

Application

Western blot

Sample

Mouse Cell lysate - whole cell (primary alveolar type II cells)

Gel Running Conditions

Non-reduced Denaturing (10%)

Loading amount

20 µg

Specification

primary alveolar type II cells

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Nov 02 2015

Application

Western blot

Sample

Human Cell lysate - whole cell (A549 cells)

Gel Running Conditions

Non-reduced Denaturing (10)

Loading amount

20 µg

Specification

A549 cells

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

DR. Christine Vohwinkel

Verified customer

Submitted Oct 29 2015

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Conjugation kits

Isotype control

Positive Controls

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Post-translationalmodifications

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (57)

Customer reviews and Q&As

IHC - Wholemount abreview for Anti-PFKFB3 antibody [EPR12594]

Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

Post-translational
modifications