Western blot analysis of extracts from Neuro2A and PC-12 cells, using MBTPS2 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
MBTPS2 Antibody detects endogenous levels of total MBTPS2 protein.Species Reactivity:
MouseSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of mouse MBTPS2. Antibodies are purified by protein A and peptide affinity chromatography.
Membrane-bound transcription factor protease site 2 (MBTPS2), also known as site-2 protease (S2P), is a zinc metalloprotease in the Golgi membrane (1,2,3). It regulates cholesterol metabolism (1,2) and unfolded protein response (UPR) (3,4). When cells are deprived of cholesterol, sterol regulatory element-binding proteins (SREBPs) move from the endoplasmic reticulum (ER) to the Golgi apparatus and are cleaved by site-1 protease (S1P) (5,6) and site-2 protease (1,6) sequentially to release the active amino-terminal domains. These amino-terminal domains of SREBPs then translocate into the nucleus to induce expression of genes for cholesterol biosynthesis. During UPR, activating transcription factor 6 (ATF6) transports from ER to Golgi apparatus and is cleaved by S1P and S2P to release a cytosolic fragment. This cytosolic fragment relocates to the nucleus and activates the UPR gene expression (7).
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