Validated using a knockout cell lineRecombinantRabMAb

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Immunocytochemistry/ Immunofluorescence - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Immunoprecipitation - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Flow Cytometry (Intracellular) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
  • Anti-Flotillin 1 antibody [EPR6041] (ab133497)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR6041] to Flotillin 1
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, IP, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

APC Carrier Free PE

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 (Intra), 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

    • WB: HEK293T, HAP1, K562, A431, Jurkat, and HeLa cell lysates; Mouse brain and kidney lysates; Rat kidney lysate. IP: K562 cell lysate. IHC-P: Human cervical carcinoma tissue. Flow Cyt (intra): Jurkat cells. ICC/IF: Jurkat 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.

    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.

Properties

Applications

The Abpromise guarantee

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 (Intra)
1/50.
WB (1)
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.
Notes
Flow Cyt (Intra)
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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    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.
  • Immunocytochemistry/ Immunofluorescence - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Immunocytochemistry/ Immunofluorescence - Anti-Flotillin 1 antibody [EPR6041] (ab133497)

    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.

  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/1000 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : FLOT1 knockout HEK293T cell lysate
    Lane 3 : HAP1 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

    Predicted band size: 47 kDa
    Observed band size: 50 kDa why is the actual band size different from the predicted?



    Lanes 1-3: Merged signal (red and green). Green - ab133497 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

     ab133497 Anti-Flotillin 1 antibody [EPR6041] was shown to specifically react with Flotillin 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267276 (knockout cell lysate ab257109) was used. Wild-type and Flotillin 1 knockout samples were subjected to SDS-PAGE. ab133497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunoprecipitation - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Immunoprecipitation - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    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
  • Flow Cytometry (Intracellular) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Flow Cytometry (Intracellular) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)

    Intracellular 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 Fluorr®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. 

     

     

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Flotillin 1 antibody [EPR6041] (ab133497)

    Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Flotillin 1 using unpurified ab133497 at a dilution of 1/100.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)

    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.

  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    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

  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    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

  • Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    Western blot - Anti-Flotillin 1 antibody [EPR6041] (ab133497)
    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?

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

References (31)

Publishing research using ab133497? Please let us know so that we can cite the reference in this datasheet.

ab133497 has been referenced in 31 publications.

  • Hayashi Y  et al. N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide ?4-Desaturase 1-Independent Mechanism. J Virol 95:e0080721 (2021). PubMed: 34106748
  • Cai S  et al. Long non-coding RNA A1BG-AS1 promotes tumorigenesis in breast cancer by sponging microRNA-485-5p and consequently increasing expression of FLOT1 expression. Hum Cell 34:1517-1531 (2021). PubMed: 34115333
  • Figueroa-Valdés AI  et al. A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells. Front Bioeng Biotechnol 9:619930 (2021). PubMed: 34124014
  • Zieren RC  et al. Extracellular vesicle isolation from human renal cancer tissue. Med Oncol 37:28 (2020). PubMed: 32172294
  • Song Z  et al. Comprehensive Proteomic Profiling of Urinary Exosomes and Identification of Potential Non-invasive Early Biomarkers of Alzheimer's Disease in 5XFAD Mouse Model. Front Genet 11:565479 (2020). PubMed: 33250918
View all Publications for this product

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Western blot abreview for Anti-Flotillin 1 antibody [EPR6041]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Bos taurus Cell lysate - other (exosomes)
Gel Running Conditions
Reduced Denaturing (4-12% Nupage Gel)
Loading amount
10 µg
Specification
exosomes
Blocking step
licor odyssey intercept buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 22°C
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Submitted Jun 03 2021

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