Western blot analysis of extracts from various cell lines using MTMR3 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
MTMR3 Antibody recognizes endogenous levels of total MTMR3 protein. This antibody also cross-reacts with a protein of unknown origin at 48 kDa.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro600 of human MTMR3 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Myotubularin-related proteins are a family of phosphatases with emerging roles in cellular signaling and membrane trafficking (1,2). MTMR3 (Myotubularin-related protein 3), also known as FYVE-DSP1, contains an amino terminal pleckstrin homology (PH) domain and a carboxyl terminal FYVE domain. MTMR3 was first reported as a dual-specific phosphatase, having phosphatase activity toward phosphorylated serine, threonine, and tyrosine residues (3). Subsequent research studies reported that MTMR3 has phosphatase activity toward phosphoinositides, including phosphatadylinositol-3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) (4). Accumulation of PI3P by the class III phosphoinositide 3-kinase Vps34 is a key element in autophagosome formation (5). Inhibition of PI3P by MTMR3 can play an important role in suppressing autophagsome formation (6).
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