Western blot analysis of extracts from various cell lines using CD9 (D8O1A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human CD9 protein (hCD9-Myc/DDK; +), using CD9 (D8O1A) Rabbit mAb.
|MW (kDa)||22, 24, 35|
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.
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
CD9 (D8O1A) Rabbit mAb recognizes endogenous levels of total CD9 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val178 of human CD9 protein.
The CD9 antigen belongs to the tetraspanin family of cell surface glycoproteins, and is characterized by four transmembrane domains, one short extracellular domain (ECL1), and one long extracellular domain (ECL2). Tetraspanins interact with a variety of cell surface proteins and intracellular signaling molecules in specialized tetraspanin-enriched microdomains (TEMs), where they mediate a range of processes including adhesion, motility, membrane organization, and signal transduction (1). Research studies demonstrate that CD9 expression on the egg is required for gamete fusion during fertilization (2-4). CD9 was also shown to play a role in dendritic cell migration, megakaryocyte differentiation, and homing of cord blood CD34+ hematopoietic progenitors to the bone marrow (5-7). In addition, down regulation of CD9 expression is associated with poor prognosis and progression of several types of cancer (8-10). Additional research identified CD9 as an abundant component of exosomes, and may play some role in the fusion of these secreted membrane vesicles with recipient cells (11).
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