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  1. Link

    pfkfb3-antibody-epr12594-ab181861.pdf

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Cardiovascular Hypoxia Hypoxia-Regulated
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-PFKFB3 antibody [EPR12594] (ab181861)

  • Datasheet
  • SDS
  • Certificate of Compliance
Reviews (6) Submit a question References (44)

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Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunocytochemistry/ Immunofluorescence - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Western blot - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Flow Cytometry (Intracellular) - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunoprecipitation - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • Immunocytochemistry/ Immunofluorescence - Anti-PFKFB3 antibody [EPR12594] (ab181861)
  • 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

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free

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Overview

  • 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.

Properties

  • 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

    • Cardiovascular
    • Hypoxia
    • Hypoxia-Regulated
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Neuroscience
    • Neurology process
    • Metabolism
    • Cancer
    • Cancer Metabolism
    • Metabolic signaling pathway
    • Metabolism of carbohydrates
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Carbohydrate metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia

Associated products

  • Alternative Versions

    • Alexa Fluor® 647 Anti-PFKFB3 antibody [EPR12594] (ab203651)
    • Alexa Fluor® 488 Anti-PFKFB3 antibody [EPR12594] (ab203984)
    • Anti-PFKFB3 antibody [EPR12594] - BSA and Azide free (ab218121)
    • Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] - Low endotoxin, Azide free (ab224262)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • HeLa whole cell lysate (ab150035)
    • HeLa whole cell lysate (ab29545)
    • Jurkat whole cell lysate (ab30128)
    • Jurkat whole cell lysate (ab7899)

Applications

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.

Target

  • 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

    Images

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

    Protocols

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

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (44)

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

    ab181861 has been referenced in 44 publications.

    • Jin X  et al. Long non-coding RNA MSC-AS1 facilitates the proliferation and glycolysis of gastric cancer cells by regulating PFKFB3 expression. Int J Med Sci 18:546-554 (2021). PubMed: 33390824
    • Yu Y  et al. Glucose metabolism involved in PD-L1-mediated immune escape in the malignant kidney tumour microenvironment. Cell Death Discov 7:15 (2021). PubMed: 33462221
    • Gong J  et al. The pentose phosphate pathway mediates hyperoxia-induced lung vascular dysgenesis and alveolar simplification in neonates. JCI Insight 6:N/A (2021). PubMed: 33497360
    • Wang F  et al. A positive feedback loop between TAZ and miR-942-3p modulates proliferation, angiogenesis, epithelial-mesenchymal transition process, glycometabolism and ROS homeostasis in human bladder cancer. J Exp Clin Cancer Res 40:44 (2021). PubMed: 33499877
    • Jiang L  et al. Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence. Nat Commun 12:879 (2021). PubMed: 33563986
    View all Publications for this product

    Customer reviews and Q&As

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    1-6 of 6 Abreviews or Q&A

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

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    Abreviews
    Abreviews
    abreview image
    Application
    IHC - Wholemount
    Sample
    Mouse Tissue (Lung)
    Specification
    Lung
    Read More
    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

    Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    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

    Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    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

    Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    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

    Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    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

    Western blot abreview for Anti-PFKFB3 antibody [EPR12594]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    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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