Western blot analysis of extracts from SJSA-1 and COLO 205 cells using CD248 Antibody (upper), and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, COLO 205 cells are low or negative for CD248 expression. Some lower bands can be apparent on the western blot, appearing beneath the CD248 band. We believe these are incompletely post-translationally modified forms of CD248.
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
CD248 Antibody recognizes endogenous levels of total CD248 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro403 of human CD248 protein. Antibodies are purified by protein A and peptide affinity chromatography.
CD248, also known as Endosialin and TEM1, is a stromal cell marker expressed on activated mesenchymal cells including fibroblasts and pericytes. Not normally detectable in most adult tissues, it is highly expressed in lymphoid tissues during development, and in disease states where increased stromal cell proliferation and migration are evident (1-3). CD248 is known to be upregulated in breast cancer, brain tumors (4-6), and other malignancies including sarcoma (12), and it has been implicated in sprouting angiogenesis and vasculogenesis (7). It is thought that the CD248 gene is upregulated in response to hypoxic conditions in the tumor environment through HIF2 activation (8). Interestingly, CD248 is found to be highly expressed in activated fibroblasts. In liver fibrosis for example, CD248 marks the Hepatic Stellate Cells, the activated cells responsible for matrix production (9,10). In kidney fibrosis, CD248 marks the key effector cells within the fibrotic stroma including pericytes, myofibroblasts, and stromal fibroblasts (11), and in Idiopathic Pulmonary Fibrosis, CD248 may be a marker for severity of disease (12). CD248 has become a target of interest for pharmaceutical intervention in a wide scope of diseases (13).
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