Western blot analysis of extracts from various cell lines using Syntaxin 5 Antibody.
|REACTIVITY||H M R|
|MW (kDa)||35, 42|
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 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
Syntaxin 5 Antibody recognizes endogenous levels of total syntaxin 5 protein. This antibody also cross-reacts with an unidentified protein of 120 kDa.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly165 of human syntaxin 5 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The membrane protein syntaxin 5 (STX5) is a key component of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complexes that regulate cellular protein transport, vesicle docking, and membrane fusion (1). Syntaxin 5 protein is found as a 42 kDa ("long") protein localized to the Golgi complex and endoplasmic reticulum, and a “short” 35 kDa isoform localized primarily to the Golgi (2,3). Formation of the syntaxin 5 SNARE complex, which also includes proteins Sec22B, Bet1, GOSR1, GOSR2, and Ykt6, allows for regulation of ER-to-Golgi transport, intra-Golgi transport, and endosome-to-Golgi retrograde transport (4-6). Research studies indicate that the syntaxin 5 SNARE complex also plays an essential role in autophagy following autophagosome formation. Intracellular protein transport mediated by the syntaxin 5 complex is required for transport and localized activity of lysosomal proteases. The experimental reduction or deletion of syntaxin 5 complex components results in non-functional lysosomes and accumulation of autophagosomes (7).
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