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Full length native protein (purified) corresponding to Human Collagen I aa 1-1464.
Database link: P02452
It is often extremely difficult to generate antibodies with specificities to collagens due to the uninterrupted "Glycine-X-Y" triplet repeat that is a necessary part of the triple helical structure. The development of type specific antibodies is dependent on NON-DENATURED three-dimensional epitopes - this may result in diminished reactivity of some antibodies with denatured collagen or formalin-fixed, paraffin embedded tissues. Anti-Collagen antibodies have been used for indirect trapping ELISA for quantitation of antigen in serum using a standard curve, for immunoprecipitation and for native (non-denaturing, non-dissociating) PAGE and western blotting for highly sensitive qualitative analysis.
Our Abpromise guarantee covers the use of ab34710 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||1/50 - 1/200.|
|Indirect ELISA||1/5000 - 1/50000.|
|WB||1/5000 - 1/10000.
This product is not recommended for use under denaturing conditions in WB, IP, and ELISA. We would suggest testing it under native conditions. Denaturing and reducing conditions will greatly diminish reactivity and selectivity of this antibody. Abcam does not test ab34710 with endogenous samples in WB. We do recommend to look at the guidelines for blotting large proteins
Customers have been successful using ab34710 in this application, please see references below (Tillgren V et al. J Biol Chem 290:918-25; 2015).
|IHC-P||Use a concentration of 5 - 10 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|ELISA||1/5000 - 1/50000.|
IHC image of Collagen I staining in normal human placenta formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab34710, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Lane 1: Human Collagen Type 1. 50 ng per lane.
Other Band(s): Collagen Type I splice variants and isoforms.
ab34710 staining mouse kidney sections (ab4606) by IHC-Fr. Kidney tissue was cryoprotected with 30% sucrose, sectioned using a cryostat at 10 microns and mounted onto slides. After drying overnight in fume hood, sections were fixed in 4% formalin in PBS for 10 minutes. Blocking was performed with 1% BSA for 10 minutes at 21°C. Staining with ab34710 at a 1/100 dilution in TBS/BSA with 0.02% azide was performed for 2h at 21°C. A conjugated goat anti-rabbit 594 polyclonal antibody at 1/1000 was used as the secondary antibody.
ab34710 staining Collagen I in horse bronchial fibroblast cells by Immunocytochemistry. Cells were fixed with acetone and blocking with 3% BSA was performed for 1 hour 30 minutes at 40°C. Samples were incubated with primary antibody (1/500: in 3% BSA/ PBS) for 12 hours at 4°C. An FITC-conjugated goat polyclonal to rabbit IgG was used at dilution at 1/160 as secondary antibody.
ab34710 staining Collagen I in Mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2.5% horse serum for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500 in PBS 1X + 3% of 2.5% horse serum) for 12 hours at 4°C. An undiluted HRP-conjugated Horse anti-rabbit polyclonal was used as the secondary antibody.
ab34710 staining Collagen I in Pig lung tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with 10% buffered formalin and blocked with 5% serum for 1 hour at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500) for 12 hours at 4°C. A Cy3-conjugated Donkey anti-rabbit polyclonal (1/200) was used as the secondary antibody.
Immunohistochemical analysis of formaldehyde-fixed frozen bovine tendon sections, labelling collagen I with ab34710 at a dilution of 1/500 for 2 hours at 21°C. 1% BSA was used as a blocking agent and incubated for 10 minutes at 21°C. Secondary used was a Goat anti-Rabbit Alexa Fluor® 594 conjugate used at 1/1,000.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"