Western blot analysis of extracts from RD and ACHN cells using COL3A1 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, ACHN cells are low or negative for COL3A1 expression.
Immunoprecipitation of COL3A1 protein from RD cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is COL3A1 Antibody. Western blot analysis was performed using COL3A1 Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
COL3A1 Antibody recognizes endogenous levels of total COL3A1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro1218 of human COL3A1 protein. Antibodies are purified by peptide affinity chromatography.
The Extracellular Matrix (ECM) is a complex network of macromolecules that provides structural tissue support to cells in the basement membrane and interstitial matrix. It is composed of many molecules including proteins, glycoproteins, proteoglycans, and polysaccharides (1,2). One of the major proteins that comprises the ECM, and the human body, is collagen. Collagens are a large family of proteins. They are trimeric molecules comprised of three alpha polypeptide chains that form a triple helix structure that is characteristic of all collagens (3). The large family of collagens is divided into three sub groups: the fibrillar collagens, the non-fibril forming collagens, and the fibril-associated collagens. These sub groups differ in their structure and supramolecular assembly (3).
COL3A1 (Collagen 3 alpha 1) is a major fibrillar collagen comprised of three identical alpha-1 chains. It is present in most soft tissues along with COL1A1, and is particularly high in tissues exhibiting elastic properties, such as the cardiac arterial wall and skin (4). Heterozygous mutations in the COL3A1 gene that cause missense mutation of a critical glycine residue in the triple helical domain of the alpha-1 chain result in vascular Ehlers-Danlos syndrome (vEDS). This mutation interferes with the ability of the alpha-1 chain to form collagen fibrils and thus disrupts macromolecular assembly of collagen fibers. vEDS is a severe and life-threatening disease as patients have a propensity for rupture of large arteries (4).
Increased amounts of type III COL3A1 are found in many fibrotic conditions such as lung, liver, kidney fibrosis, and systemic sclerosis (5,6).
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