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Full length native protein (purified) corresponding to Collagen III aa 1-1466.
Database link: P02461
For more protocol tips, please see: http://www.abcam.com/protocols/collagen
At least 11 genetically distinct gene products are collectively referred to as 'collagen types' or other proteins and proteoglycans of the extracellular matrix. In humans, collagens are composed of about 20 unique protein chains which under go various types of post-translational modifications and are ultimately assembled into a triple helix. This results in great diversity between collagen types. Collagens are highly conserved throughout evolution and are characterized by an uninterrupted "Glycine-X-Y" triplet repeat that is a necessary part of the triple helical structure. For these reasons it is often extremely difficult to generate antibodies with specificities to collagens. The development of type specific antibodies is dependent on NON-DENATURED three-dimensional epitopes. This preparation results in a native conformation of the protein.
Our Abpromise guarantee covers the use of ab7778 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ICC/IF||Use at an assay dependent concentration. PubMed: 18385800|
|IF||Use at an assay dependent concentration. PubMed: 28135282|
|ICC||1/100. PubMed: 19036760|
|ELISA||1/1000 - 1/8000. This product was assayed against 1.0 ug of Collagen III in a direct ELISA using Peroxidase conjugated Goat anti-Rabbit and ABTS (2,2'-azino-bis-[3- ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at room temperature. This product can also be used with Biotin Conjugated Anti-Collagen III (ab6580) in a sandwich ELISA.|
|IHC-Fr||1/50 - 1/200.|
|IP||1/5000 - 1/10000.|
|WB||1/5000 - 1/10000. Predicted molecular weight: 138 kDa.|
ab7778 at 1:400 (45 min RT) showed strong staining in FFPE sections of human skin(left, dermis) with moderate to strong red staining and testis (right) where strong staining was observed within connective tissue between seminiferous tubules. The antibody showed strong extracellular staining within connective tissues across many organs with minimal background staining. Slides were steamed in 0.01 M sodium citrate buffer, pH 6.0 (ab64214) at 99-100°C - 20 minutes for antigen retrieval.
Ab53088 staining Human skin (ab30166). Staining is localised to the extracellular matrix.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the DAKO 3-in-1 antigen retrieval buffer citrate pH 6.0 (ab64214) in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
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