For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
ADAMTS1 (D5G4Z) Rabbit mAb recognizes endogenous levels of total ADAMTS1 protein. This antibody does not cross-react with other ADAM or ADAMTS proteins.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg187 of human ADAMTS1 protein.
A Disintegrin and Metalloprotease with Thrombospondin Motifs (ADAMTS) proteins comprise a large family of secreted zinc metalloproteases that play important roles in various processes, including organogenesis, hemostasis, and angiogenesis (1,2). ADAMTS proteases show structural similarity to ADAM proteases, but are further defined by the presence of repeated carboxy-terminal motifs homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (3). Functions ascribed to ADAMTS proteases include regulating the extracellular bioavailability of cytokines and growth factors (4, 5), regulating cell adhesion to the extracellular matrix (ECM), and remodeling of the ECM (6).
ADAMTS1 has been shown to possess potent anti-angiogenic activity in vitro (2) and is reportedly dysregulated in a number of cancer subtypes (7). Functional in vivo studies in an ADAMTS1 knockout mouse model suggested that ADAMTS1 promotes metastatic invasion of breast carcinoma cells (8). These studies showed a reduced tumor burden in ADAMTS1 knockout mice, which was linked to increased cytotoxic immune cell invasion and reduced tumor cell survival (8).
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