Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Flotillin 1 antibody [EPR6041] (ab133497)

Knockout Tested Rabbit recombinant monoclonal Flotillin 1 antibody [EPR6041]. Validated in WB, IP, IHC, Flow Cyt, ICC/IF and tested in Mouse, Rat, Human. Cited in 8 publication(s).

Overview

  • Product name
    Anti-Flotillin 1 antibody [EPR6041]
    See all Flotillin 1 primary antibodies
  • Description
    Rabbit monoclonal [EPR6041] to Flotillin 1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, WB, IHC-P, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Flotillin 1 aa 400-500 (C terminal). The exact sequence is proprietary.
    Database link: O75955

  • Positive control
    • K562, A431, Jurkat, and HeLa cell lysates, Human colon tissue
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab133497 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 1/50.
WB 1/10000 - 1/50000. Detects a band of approximately 48 kDa (predicted molecular weight: 47 kDa).
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

IP 1/10 - 1/100.
ICC/IF 1/500.

Target

Images

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: Flotillin 1 knockout HAP1 whole cell lysate (20 µg)

    Lanes 1 - 2: Merged signal (red and green). Green - ab133497 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab133497 was shown to specifically react with Flotillin 1 in wild-type HAP1 cells as signal was lost in Flotillin 1 knockout cells. Wild-type and Flotillin 1 knockout samples were subjected to SDS-PAGE. Ab133497 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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.

  • All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution (purified)

    Lane 1 : K562 cell lysate
    Lane 2 : A431 cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 47 kDa
    Observed band size: 48 kDa
    why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab133497 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
  • Immunofluorescence staining of Jurkat cells with purified ab133497 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab133497 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

  • Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling Flotillin 1 with purified ab133497 at 1/50 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.

  • All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution (purified)

    Lane 1 : mouse brain lysate
    Lane 2 : mouse kidney lysate
    Lane 3 : rat kidney lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L)

    Predicted band size: 47 kDa
    Observed band size: 48 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution

    Lane 1 : K562 cell lysate
    Lane 2 : A431 cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : HeLa cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size: 47 kDa
    Observed band size: 48 kDa why is the actual band size different from the predicted?

  • ab133497 (purified) at 1/60 immunoprecipitating Flotillin 1 in 10 μg K562 (Lanes 1 and 2, observed at 48 kDa). Lane 3 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST
  • Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Flotillin 1 using unpurified ab133497 at a dilution of 1/100.

References

This product has been referenced in:
  • Reisinger SN  et al. Flotillin-1 interacts with the serotonin transporter and modulates chronic corticosterone response. Genes Brain Behav N/A:e12482 (2018). Read more (PubMed: 29667320) »
  • Wang T  et al. Increasing circulating exosomes-carrying TRPC5 predicts chemoresistance in metastatic breast cancer patients. Cancer Sci 108:448-454 (2017). Read more (PubMed: 28032400) »
See all 8 Publications for this product

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