Overview

  • Product name
    Anti-Cortactin antibody [4F11]
    See all Cortactin primary antibodies
  • Description
    Mouse monoclonal [4F11] to Cortactin
  • Host species
    Mouse
  • Specificity
    The clone number has been updated from (0.T.21) to (4F11) both clone numbers name the same antibody clone.
  • Tested applications
    Suitable for: ICC/IF, WB, IP, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Hamster, Cow, Human
  • Immunogen

    Mixture of affinity purified tyrosine phosphoproteins from chick embryo fibroblasts expressing activated pp60 src.

  • Positive control
    • WB: 3T3 and src transformed chicken fibroblast cell lysates. ICC/IF: Wildtype HAP1 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab33333 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.
WB Use a concentration of 0.5 - 2 µg/ml. Predicted molecular weight: 61 kDa. Use 20 µg per lane of 3T3 cell lysate as a positive control for mini gels.
Note: ab33333 recognizes 80kD and 85kD proteins in avian cells, but it recognizes an 80kD protein in rodent and human cells.
IP Use at 10 µg/mg of lysate.
ICC Use a concentration of 10 µg/ml.

Target

Images

  • ab33333 staining Cortactin in wild-type HAP1 cells (top panel) and CTTN knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab33333 at 1μg/ml and ab7291 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • All lanes :

    Lane 1 : HAP1 whole cell lysate
    Lane 2 : HAP1 CTTN whole cell lysate
    Lane 3 : NIH3T3 whole cell lysate
    Lane 4 : A431 whole cell lysate
    Lane 5 : PC12 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 61 kDa
    Observed band size: 80 kDa
    why is the actual band size different from the predicted?



    ab33333 was shown to specifically react with CTTN (Cortactin) in wild type HAP1 cells. No band was observed when CTTN (Cortactin) knockout samples were used. Wild-type and CTTN (Cortactin) knockout samples were subjected to SDS-PAGE. ab33333 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at a 1ug/ml concentration and 1/10000 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 1/20000 dilution for 1 hour at room temperature before imaging.

  • Anti-Cortactin antibody [4F11] (ab33333) at 1/1000 dilution + HEK293T cells at 10 µg

    Secondary
    Polyclonal goat anti-mouse HRP at 1/10000 dilution

    Predicted band size: 61 kDa



    Samples were incubated with primary antibody for 2 hours at 20ºC.

    Blocking: 5% milk for 1 hour at 20ºC.

    See Abreview

  • Anti-Cortactin antibody [4F11] (ab33333) at 1/1000 dilution + Bovine aortic endothelial whole cell lysates at 10 µg

    Secondary
    Goat anti-mouse polyclonal HRP conjugate at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 61 kDa
    Additional bands at: 80 kDa (possible post-translational modification)


    Exposure time: 5 seconds


    Blocking was with 5% milk for 1 hour at 20°C.

    See Abreview

  • ab33333 staining Cortactin (red) in Human palladin-activated fibroblasts by Immunocytochemistry/ Immunofluorescence.

    Cells were fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. Samples were incubated with primary antibody (1/500 in diluent) before incubation with a Rhodamine Red IgG (1/100).

References

This product has been referenced in:
  • Valenzuela-Iglesias A  et al. Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner. Mol Cancer Res N/A:N/A (2019). Read more (PubMed: 30655320) »
  • Ghosh S  et al. A Role for ßA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 59:AMD104-AMD113 (2018). Read more (PubMed: 30098172) »
See all 25 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Sample
African Green Monkey Cell lysate - whole cell (COS7 cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
COS7 cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Oct 16 2015

Application
Western blot
Sample
Dog Cell lysate - whole cell (Madin-Darby Canine Kidney (MDCK) epithelial cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Madin-Darby Canine Kidney (MDCK) epithelial cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Sep 29 2015

Application
Western blot
Sample
Cow Cell lysate - whole cell (Bovine aortic endothelial cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Bovine aortic endothelial cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Sep 25 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
HEK293T cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 19 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 0.5% · Temperature: 20°C
Sample
Mouse Cell (J774 macrophage)
Specification
J774 macrophage
Permeabilization
Yes - 0.05% saponin
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted May 23 2014

Answer

Thank you for contacting us. I am sorry to hear that this antibody is not providing satisfactory results.

Would you be able to provide the lot number and order number?

It may be possible that the ˜100 kDa band corresponds to the phosphorylated form of cortactin. Since the cortactin antibody you're using ab33333 is a monoclonal with one specific epitope, it may not have an epitope that encompasses every phosphorylation site (24 possible from Swiss Prot) present on cortactin to make it a true indicator of total cortactin (meaning phospho- and non-). Plus phosphoproteins run higher in gels, so it seems to fit where we may expect the band to arise.

Also, are you treating your cells to induce phosphorylation at this particular site (Y421)? Our positive control was HeLa cells treated with 150 uM H2O2. Is it known the squamous cell carcinomas endogenously express this phosphorylation? The best information I could find online was with a serum starved squamous cell carcinoma of the oropharynx. Were your cells serum starved? http://www.phosphosite.org/siteAction.do?id=2685

Lastly, if you are blocking in milk, I'd recommend switching to 5% BSA throughout the entire experiment since milk contains endogenous phosphatases.

I hope this information helps. I look forward to your reply so that we may help you further.

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