Analysis Note

ControlHuman kidney lysate

Application

ELISA: detection of native type I collagen. Capture: 2-4 μg/ml; Detection: A dilution of 10μg/mL of Collagen Type I Antibody will detect 50ng of human Collagen Type I with no reactivity with other Collagens. Higher levels of antibody may be necessary to detect lower concentrations of Collagen. Note antibody will not pair with itself.

Immunohistochemistry:unfixed frozen sections:

Cut 6 μm thick sections from frozen samples using a freezing microtome. Air-dry throughly. Rehydrate with PBS. Blocking is usually not necessary for IF studies, if using DAB, pretreatment for peroxidase is suggested and blocking 2% BSA in PBS will reduce back ground.

Dilute Collagen Type I Antibody in PBS buffer, add to the sample(s) and incubate for 60 minutes. (1:100-1:500).

Wash sections for 10 minutes in PBS, twice. Add FITC conjugated anti-mouse antibody and incubate for 60 minutes.

Wash for 10 minutes in PBS, twice.

Mount sections in glycerol/water (9:1 v/v) containing 1 mM 1,4-phenylenediamine. (for FITC stability or use Chemicon catalog numbers 5096 or 5013.

NOTE:

Preliminary studies suggest that enhanced staining of certain tissues maybe obtained by pretreatment (previous to step 2) of sections with various enzymes (eg 0.1% pepsin in 0.1M HCl, 37°C for 5 minutes).

Flow Cytometry: 1:100

Immunoblotting:1:1000 using NATIVE, non-denatured, non-reduced western blots only. Ramshaw, et.al (1988) 'Electrophoretic and electroblotting of native collagens.' Anal. Biochem. 168:82-87. Antibody will not work in traditional, reduced western blots.

Briefly, PAGE gels must be prepared for running native collagens.

A 3% (w/v) total acrylamide separating gel, containing 3.2% (w/w) bis-acrylamide as a proportion to a monomer as the crosslinking agent, in 10mM calcium lactate, pH 6.8 is polymerized between vertical glass plates by the addition of 0.05-0.1% TEMED and 0.05-.1% ammonium persulfate (added from a 10% stock solution). The cathode buffer (negative) buffer is 50mM Tris adjusted to pH 6.6 with lactic acid; the anode (positive) buffer is 0.1M lactic acid pH 2.5 {Friis, SJ et al, 1985 J Biochem Biophys Methods 10:301-306.}.

Prior to sample loading the gel is run for at least 90 minutes at 100V. Collagen samples are dissolved at 1mg/mL in either 0.1M lactic acid or 0.1M acetic acid containing 10% sucrose, and 5-10μg per lane is loaded. Methyl green is added as required to assist loading. Electrophoresis is 70V for 5 hours, room temperature.

Blotting uses 0.1M lactic acid pH 2.5, and 20V for 16 hours at room temperature with the collagens migrating toward the cathode.

Research CategoryCell Structure

This Anti-Collagen Type I Antibody, clone 5D8-G9 is validated for use in ELISA, FC, WB, IH for the detection of Collagen Type I.

Research Sub CategoryECM Proteins

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Collagens are highly conserved throughout evolution and are characterised by an uninterrupted "Glycine X Y" triplet repeat that is a necessary part of the triple helical structure. Type I collagen (95 kDa) is found in bone, cornea, skin and tendon. Mutations in the encoding gene are associated with osteogenesis imperfecta, Ehlers Danlos syndrome, and idiopathic osteoporosis. Reciprocal translocations between chromosomes 17 and 22, where this gene and the gene for Platelet-derived growth factor beta are located, are associated with a particular type of skin tumor called dermatofibrosarcoma protuberans, resulting from unregulated expression of the growth factor.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Physical form

100µg purified antibody at a concentration of 1mg/mL in 50 mM Tris Acetate, 150 mM NaCl, 0.1% Bronidox (pH 5.5).The immunoglobulin fraction has been purified by Protein A Chromatography and shown to be >95% pure by coomassie PAGE. The Collagen Type I Antibody is supplied 0.22 micron filtered.

Format: Purified

Specificity

Collagen Type I Antibody is a monoclonal IgG1 antibody which binds an epitope near the C-terminal of Type I Collagen.

Collagen Type I Antibody displays a high affinity for human, bovine and ovine Type I Collagens. There is no evidence for cross reactivity with Collagen Types III, V and VI or connective tissue protein. Antibody reacts only with native, non-denatured Collagen I

Storage and Stability

Collagen Type I Antibody is shipped, in liquid form, at ambient temperature. This material is stable for up to 6 months when stored at 2-8°C.

WARNING: The monoclonal reagent solution contains 0.1% sodium azide as a preservative. Due to potential hazards arising from the build up of this material in pipes, spent reagent should be disposed of with liberal volumes of water.