Overview

  • Product name

  • Description

    Rabbit polyclonal to PFKFB4
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ELISA, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide corresponding to Human PFKFB4 aa 266-296 (internal sequence) conjugated to keyhole limpet haemocyanin.

  • Positive control

    • IHC-P:Formalin-fixed, paraffin-embedded human hepatocarcinoma tissue and mouse brain tissue lysate. WB: Raji, Molt-4, NCI-H1299, 293, and U-87 MG whole cell lysate ICC/IF: HepG2

Properties

Applications

Our Abpromise guarantee covers the use of ab71622 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
WB
ELISA
IHC-P
ICC/IF
  • Application notes
    ELISA: 1/1000.
    IHC-P: 1/50 - 1/100.
    WB: 1/100 - 1/500. Detects a band of approximately 60 kDa and many other bands (predicted molecular weight: 54 kDa).


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    Images

    • All lanes : Anti-PFKFB4 antibody (ab71622) at 1/1000 dilution

      Lane 1 : Raji, whole cell lysate with 5% NFDM/TBST.
      Lane 2 : Molt-4 whole cell lysate with 5% NFDM/TBST.
      Lane 3 : NCI-H1299 whole cell lysate with 5% NFDM/TBST.
      Lane 4 : 293 whole cell lysate Lane with 5% NFDM/TBST.
      Lane 5 : U-87 MG whole cell lysate with 5% NFDM/TBST.

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution

      Predicted band size: 54 kDa

    • Formalin-fixed and paraffin-embedded human hepatocarcinoma tissue reacted with ab71622 at a 1:50 dilution. A peroxidase-conjugated secondary antibody was then used, followed by AEC staining.
    • Formalin-fixed and paraffin-embedded human hepatocarcinoma tissue reacted with ab71622 at 1:50 dilution. A peroxidase-conjugated secondary antibody was then used, followed by DAB staining.
    • ICC/IF image of ab71622 stained HepG2 cells. The cells were 4% PFA fixed (10mins) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab71622, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    References

    ab71622 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A

    Answer

    Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from these antibodies.

    The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality.

    Reviewing this case, I would like to offer some suggestions to help optimise the results from ab65979:

    1.) HIF alpha is a notoriously difficult target since it is degraded quickly and only really detectable during optimal hypoxia conditions. During this time is localised in the nucleus
    Since the blot shows multiple bands that are all smaller in size than HIFa, it is very likely that we see to results of degradation. I therefore suggest to optimise to hypoxia conditions.
    I also strongly recommend to use SDS in the lysis buffer to ensure that the nucleus is lysed as well to retrieve the nuclear HIFa. I suggest to use 0.1% SDS. This can be increased if necessary.

    2.) To eliminate the possibility that the background is due to the secondary antibody, I suggest to run a no primary control. By omitting the primary antibody one can be sure that all bands seen are background generated by the secondary antibody.

    3.) I also strongly suggest to use a positive control. In this case we can recommend rat brain lysate or HeLa cell lysate.

    ab71622:

    1.) PFKFB4 is a protein that is phophorylated at multiple sites. Phophogroups can add considerable weight to the calculated protein weight and I am confident that the strong band at ca 60kDa is the correct band.

    2.) The background can be reduced by using less antibody and less protein loaded on the gel. Any antibody if used to concentrated will produce high background.

    3.) I can also recommend to use a positive control. I suggest HepG2 or NIH3T3 cells.

    4.) In general I cannot recommend to mix blocking agents. Either use milk or BSA. The blocking agent also can be optimised. The signal strength and the background can be influenced by the choice of blocking agent.

    ab110025:

    1.) Mitochondrial Pyruvate dehydrogenase kinase 1 gives relatively a weak band in these results. I therefore suggest to use a positive control such as HeLa cells, Jurkat or HepG2 cells.

    2.) Since the general background increases strongly after a short exposure of only 5 seconds, I suggest to run a no primary control to exclude that the background is generated by the secondary antibody.

    3.) To have the most efficient lysis of the Mitochondrion matrix where Mitochondrial Pyruvate dehydrogenase kinase 1 is localised I recommend to use 0.1% SDS in the lysis buffer.

    We are happy to offer this technical support. In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

    I hope this information is helpful, thank you and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.

    Read More
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HCT116)
    Loading amount
    25 µg
    Specification
    HCT116
    Gel Running Conditions
    Reduced Denaturing (10%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Nov 23 2010

    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (NIH 3T3 cells)
    Loading amount
    25 µg
    Specification
    NIH 3T3 cells
    Gel Running Conditions
    Reduced Denaturing (10%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Nov 23 2010

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