Western blot analysis of extracts from various cell lines using CD2 (D5L3B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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
CD2 (D5L3B) Rabbit mAb recognizes endogenous levels of total CD2 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala36 of human CD2 protein.
CD2 is a transmembrane glycoprotein expressed early in thymocyte development and present on most circulating T cells (1, 2). CD2 plays a role in T cell adhesion through binding to its ligand CD58 (LFA-3) (3). Stimulation of CD2 also leads to T cell activation and proliferation (2). T cells from mice deficient in both CD2 and CD28 have severe defects in T cell activation and function, while T cells deficient in either CD2 or CD28 are still capable of mounting a response, suggesting that CD2 and CD28 may have overlapping functions and may be able to compensate for each other (4). In addition, engagement of CD2 and CD58 was recently demonstrated to be the primary costimulatory signal in T cells that lack CD28 (5). CD2 expression also distinguishes a subset of plasmacytoid dendritic cells found in tumors and tonsils that express lysozyme, higher levels of IL-12 p40, and higher levels of CD80 (6).
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