Western blot analysis of extracts from various cell lines using Tissue Factor/CD142 Antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human tissue factor protein (hTF-Myc/DDK; +), using Tissue Factor/CD142 Antibody (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from BxPC-3 cells, PANC-1 cells, and Mia PaCa2 cells using Tissue Factor/CD142 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, Tissue Factor/CD142 protein is not detected in either Mia PaCa2 cells or PANC-1 cells.
Western blot analysis of extracts from MDA-MB-231 cells, untreated (-) or treated with PNGase F (+), using Tissue Factor/CD142 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Tissue Factor/CD142 Antibody recognizes endogenous levels of total Tissue Factor/CD142 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu123 of human Tissue Factor/CD142 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Tissue Factor (TF)/CD142 (Coagulation factor III/Thromboplastin) is a type-I transmembrane glycoprotein that serves as the cell surface receptor and cofactor for blood coagulation factors VII and VIIa, and thus plays a central role in hemostasis and thrombosis (1). The TF:VIIa receptor-ligand complex is widely recognized as the initiator of the extrinsic blood coagulation protease cascade, which ultimately leads to the generation of fibrin and thrombin (1). A member of the type-II cytokine receptor superfamily, TF has also been shown to engage the PI3K (2) and MAPK (3) signaling cascades upon binding to factor VIIa in order to drive cellular responses such as cell migration, growth, and proliferation. Although the function of TF under physiologic conditions is to coordinate blood clotting in response to tissue damage, TF is implicated in pathologic conditions such as tumorigenesis. Indeed, TF is aberrantly expressed in colorectal cancer, breast cancer, pancreatic cancer, and glioblastoma multiforme (4). It has been shown to promote tumor angiogenesis, tumor growth, metastasis, and venous thrombosis (5). Given that TF overexpression is associated with numerous types of solid tumors, it has garnered much attention as a potential therapeutic target.
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