Western blot analysis of extracts from various cell lines using Rab27B Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of Rab27B expression in SK-MEL-5 cell extracts is consistent with molecular and proteomic expression profiling data, confirming specificity of the antibody.
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
Rab27B Antibody recognizes endogenous levels of total Rab27B protein. Overexpression analysis confirms that this antibody does not detect Rab27A.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human Rab27B protein. Antibodies are purified by protein A and peptide affinity chromatography.
Rab27 proteins are members of the Ras superfamily of small Rab GTPases associated with the process of exocytosis (1-2), and in particular the exosome secretion pathway (3-5). There are two Rab27 isoforms, Rab27A and Rab27B, which are encoded by separate genes and which exhibit distinct subcellular localization and functions. Rab27A colocalizes to CD63+ vesicles, whereas Rab27B is observed in perinuclear vesicles. Targeted knockdown studies suggest that the isoforms control distinct steps of the exosome secretion pathway (6). Rab27A participates in docking and membrane fusion from multivesicular endosomes (MVEs) to exosomes, whereas Rab27B mediates the transfer of membrane vesicles from the trans-Golgi network to MVEs. It has been suggested that Rab27A and Rab27B may have importance in exosome secretion by cancer cells (7).
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