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corresponding to Human Sonic Hedgehog.
(Peptide available as
This product is a recombinant rabbit monoclonal antibody.
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc, a Thermo Fisher Scientific Company. The Alexa Fluor® dye included in this product is provided under an intellectual property license from Life Technologies Corporation. As this product contains the Alexa Fluor® dye, the purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). As this product contains the Alexa Fluor® dye the sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: in manufacturing; (ii) to provide a service, information, or data in return for payment (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are sold for use in research. For information on purchasing a license to use products containing Alexa Fluor® dyes for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or email@example.com
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.
Our Abpromise guarantee covers the use of ab53281 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/250 - 1/500.|
|WB||1/1000 - 1/10000. Detects a band of approximately 51 kDa (predicted molecular weight: 51 kDa).Can be blocked with Sonic Hedgehog peptide (ab203144).|
|Flow Cyt||1/10 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).
For unpurified use at 1/100.
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/250 dilution (0.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreatic carcinoma tissue sections labeling Sonic Hedgehog with purified ab53281 at 1/2000 dilution (0.097 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.
Blocking and diluting buffer: 5% NFDM/TBST.
50kDa: Full-length SHH; 27KDa: C-terminal product
Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue).
Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Sonic Hedgehog with purified ab53281 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised by 0.1% tritonX-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).
Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Sonic Hedgehog with unpurified ab53281.
Blocking and diluting buffer: 5% NFDM /TBST .
27KDa: C-terminal product.
Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Sonic Hedgehog with unpurified ab53281 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
Immunohistochemical analysis of Formalin fixed paraffin-embedded human kidney cancerous tissue labeling Sonic Hedgehog with unpurified ab53281 at 1/100 dilution.
Immunohistochemical analysis of Formalin fixed paraffin embedded human ovarian cancer tissue, staining Sonic Hedgehog with unpurified ab53281.
Antigen retrieval was carried out in citrate buffer using a pressure cooker for 40 minutes. Sections were blocked with blocking agent before incubating with primary antibody (1/2000) for 90 minutes at room temperature. Staining was detected using DAB.
Overlay histogram showing HepG2 cells stained with unpurified ab53281 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab53281, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.