Overview

  • Product name

  • Description

    Rabbit polyclonal to ARF6
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, IHC-P, WBmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Cow, Pig, Xenopus laevis
  • Immunogen

    Synthetic peptide corresponding to ARF6 aa 150 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab86625)

  • Positive control

    • This antibody gave a positive signal in the following whole cell lysates: HeLa whole cell, HEK293 whole cell, HepG2 whole cell, Human Liver Tissue

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab77581 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
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 18 kDa (predicted molecular weight: 20 kDa).

Target

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: ARF6 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: HepG2 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab77581 observed at 18 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab77581 was shown to recognize ARF6 when ARF6 knockout samples were used, along with additional cross-reactive bands. Wild-type and ARF6 knockout samples were subjected to SDS-PAGE. ab77581 and ab8245 (loading control to GAPDH) were diluted 1 μg/mL and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

  • IHC image of ARF6 staining in human liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab77581, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • All lanes : Anti-ARF6 antibody (ab77581) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 4 : Human liver tissue lysate - total protein (ab29889)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 20 kDa
    Observed band size: 18 kDa
    why is the actual band size different from the predicted?



    We hypothesize that the 18 kDa band represents the mature form of ARF6.
  • ICC/IF image of ab77581 stained HeLa cells. The cells were 4% PFA fixed (10 min) 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 (ab77581, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa cells at 1µg/ml.
  • All lanes : Anti-ARF6 antibody (ab77581) at 1 µg/ml

    Lane 1 : Lysates prepared from Mouse liver tissue.
    Lane 2 : Lysates prepared from Mouse lung tissue.
    Lane 3 : Lysates prepared from Rat liver tissue.
    Lane 4 : Lysates prepared from Rat lung tissue.

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP-conjugated Pig polyclonal to rabbit IgG at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 20 kDa
    Observed band size: 21 kDa why is the actual band size different from the predicted?


    Exposure time: 2 minutes

    See Abreview

  • Immunohistochemical analysis of mouse Salivary Gland staining ARF6 with ab77581 at 1/50 dilution. An undiluted HRP conjugated Polyclonal anti rabbit was used as a secondary. Blocked with PB ab64226.

    See Abreview

References

This product has been referenced in:

  • Pua HH  et al. Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses. Cell Rep 26:933-944.e4 (2019). Read more (PubMed: 30673615) »
  • Patel GK  et al. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Sci Rep 9:5335 (2019). Read more (PubMed: 30926864) »
See all 17 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Salivary Gland)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 9.0 EDTA
Permeabilization
No
Specification
Salivary Gland
Blocking step
PB ab64226 as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 10 2017

Answer

Merci pour votre requête.

Le laboratoire a testé cet anticorps sur en gel migré BisTris 10% ou 4-12%, en tampon MES. La membrane était bloqué ensuite en 5%BSA en 0.1TBST. L'incubation de l'anticorps s'est fait à une dilution de 1/1000 dans le tampon de blocage pour la nuit à 4°C. Les rinçages se sont fait enTBS Tween 0.1%.

J'espère que ces informations vous sont utiles. Si jamais vous avez des problèmes avec un de nos produits, veuillez ne pas hésiter à nous recontacter avec plus de détails. Le service technique vous donne volontiers des conseils et nous avons aussi une garantie sur nos produits de fonctionner tel que décrit sur la fiche technique (https://www.abcam.com/abpromise).

N'hésitez pas à nous recontacter si vous avez besoin de plus de renseignements.

Read More
Application
Western blot
Sample
Mouse Tissue lysate - whole (Ms Liver, Ms Lung, Rat Liver, Rat Lung)
Loading amount
20 µg
Specification
Ms Liver, Ms Lung, Rat Liver, Rat Lung
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Mr. Antibody Solutions

Verified customer

Submitted Jan 12 2010

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