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
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.
BST2 (D5V5Z) Rabbit mAb recognizes endogenous levels of total BST2 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val134 of human BST2 protein.
BST2 (CD317, Tetherin, HM1.24) is a type II transmembrane glycoprotein functioning as a major mediator of the innate immune defense against the dissemination of enveloped viruses by tethering viron on cell surface (1). BST2 has a N-terminal cytoplasmic tail for entocytosis and cytoskelatal signaling, a transmembrane domain, an extracellular domain containing putative disulfide bonds and coiled coil region for forming homodimer, and a C-terminal GPI domain for membran anchoring (2,3). Both the transmembrane domain and the GPI domain can insert either to the cell membrane or the viral envelope membrane and hold them together to prevent viral release. Virus counteracts BST2 by encoding viral protein as antagonist. These viral proteins interact directly with BST2 to either enhance BST2 endocytosis/lysosomal degradation (such as Vpu) or prevent BST2 secretion pathway by sequestering the protein in endosome (2, 3). BST2 is overexpressed in gastrointestinal cancers, breast cancer, lung cancer and multiple myeloma (4-7). BST2 monoclonal antibody targeting myeloma or lung cancer cells induces celllular cytotoxicity and cell death (ADCC, antibody-dependent cell-mediated cytotoxicity). Thus BST2 serves as a potential target for tumor immunotherapy.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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