Overview

  • Product nameAnti-Smoothened antibody
    See all Smoothened primary antibodies
  • Description
    Rabbit polyclonal to Smoothened
  • Tested applicationsSuitable for: WB, ICC/IF, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Horse, Chicken, Pufferfish (Fugu), Zebrafish
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 750 to the C-terminus of Human Smoothened.

    (Peptide available as ab39344.)

  • Positive control
    • This antibody gave a positive signal against Smoothened partial recombinant (tagged) protein.

Properties

Applications

Our Abpromise guarantee covers the use of ab38686 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 Use a concentration of 1 µg/ml. Predicted molecular weight: 86 kDa.
ICC/IF Use a concentration of 5 µg/ml.
IHC-FoFr 1/3000.

Target

Anti-Smoothened antibody images

  • Anti-Smoothened antibody (ab38686) at 1 µg/ml + Human Smoothened Partial Recombinant Protein (Tagged) at 0.1 µg

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

    Performed under reducing conditions.

    Predicted band size : 86 kDa
    Observed band size : 41 kDa (why is the actual band size different from the predicted?)
    ab38686 was tested against Human Smoothened Partial Recombinant Protein (Tagged) predicted to run at 41 kDa.
  • ICC/IF image of ab38686 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 (ab38686, 5µg/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).
  • Immunohistochemistical detection of Smoothened protein with antibody ab38686 on rat brain PFA-perfusion fixed frozen sections. Primary antibody ab38686 was diluted 1/3000 and incubated for 18 hours @ 20°C in PBS + 0.3 % TritonX100. Secondary antibody: goat anti-rabbit Alexa Fluor 488® (1/1000).  This antibody was used on (30um) free-floating sections of rat brain. The antibody produced some staining in the whole CNS, in glial cells as well as some diffuse staining. No staining was observed in the dentate gyrus although this was expected as Rajendran et al (Neurosci Lett. 2009 April 10 453(3): 190–194) showed the presence of Smoothened mRNA in this structure. However, literaure on the protein expression is very limited; it is possible that the protein might be expressed in gial cells too. The pictures submitted show the staining at the level of the striatum (top panel). ab38686 stains some neuron-like cells and some glial cel

    See Abreview


  • Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 86 kDa


    Exposure time : 10 seconds

    NIH-3T3 cells were incubated at 37°C for 7h with vehicle control (0 µM) and 5 µM of jervine (ab120815) in DMSO. Decreased expression of smoothened (ab38686) in NIH-3T3 cells correlates with an increase in jervine concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab38686 at 1 µg /ml and ab8227 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

References for Anti-Smoothened antibody (ab38686)

This product has been referenced in:
  • Jung B  et al. Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation. PLoS One 11:e0149477 (2016). WB, IP, ICC/IF . Read more (PubMed: 26901434) »
  • Chen Q  et al. Down-regulation of Gli transcription factor leads to the inhibition of migration and invasion of ovarian cancer cells via integrin ß4-mediated FAK signaling. PLoS One 9:e88386 (2014). WB ; Human . Read more (PubMed: 24533083) »

See all 14 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (primary osteoblasts)
Permeabilization Yes - 0.05% Triton
Specification primary osteoblasts
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative Paraformaldehyde
Username

Dr. Tina Yuan

Verified customer

Submitted Dec 18 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (Mensenchymal stem cells)
Gel Running Conditions Reduced Denaturing
Loading amount 30 µg
Specification Mensenchymal stem cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. Tina Yuan

Verified customer

Submitted Dec 16 2015

Application Western blot
Sample Human Cell lysate - other (fibroblasts - Cyto fraction)
Gel Running Conditions Reduced Denaturing
Loading amount 50 µg
Specification fibroblasts - Cyto fraction
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Apr 17 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample Rat Cell (neural stem cells)
Specification neural stem cells
Permeabilization Yes - 0.2% Tween
Fixative Methanol
Username

Dr. Christine Heberden

Verified customer

Submitted Jul 31 2013


The protein fragment is available as ab114221 in our catalog now.
http://www.abcam.com/ab114221.html

It runs at 42 kDa since it is only a fragment not the full length protein.


We have clarified the band size for the WB image with jervine treatment:
The band size of 42 kDa on that WB for ab38686 is a typo and it's actually running at around 86 kDa - as expected. This is nicely in line with the predicted MW and matche...

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Thank you for contacting us. The WB image shown on the datasheet is actually from partial recombinant protein, so the size is smaller. The predicted size would still be 86kD for human and 87 kD for mouse in samples detecting endogenous protein.
I h...

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Thank you for your enquiry regarding ab4058 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

It is very clear from the attached images that the...

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I am very sorry to hear that you have been experiencing problems using our products (ab4058, ab38686, ab72130, ab80645) in the application that you wish.

As our Abpromise indicates, in the event that a product is not functioning in the applic...

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The only other non rabbit antibody against CEP290 is ab77479, which is a goat polyclonal:

http://www.abcam.com/CEP290-antibody-ab77479.html



This is one of our fast track antibodies, which means it has not been fully characte...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"