Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with CCCP (100μM, 2hr; +), using phospho-MFF (Ser146) Antibody (upper) or total MFF antibody (lower).
Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with CCCP (100uM, 2hr; +), using Phospho-MFF (Ser146) Antibody. The phospho-specificity of the antibody was verified by treating the membrane with (+) or without (-) calf intestinal phosphatase (CIP) after protein transfer.
|REACTIVITY||H M R|
|MW (kDa)||25, 27|
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
Phospho-MFF (Ser146) Antibody recognizes endogenous levels of MFF protein only when phosphorylated at Ser146. This antibody has been shown to react with phosphorylated Ser146 of human MFF isoforms 2, 4, and 5 but not phosphorylated Ser172 of human MFF isoform 1 (7). This antibody cross-reacts with a 140kDa protein of unknown identity.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser146 of human MFF isoform 2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Mitochondrial fission factor (MFF) is a tail-anchored protein that resides within the outer mitochondrial membrane and is part of the mitochondrial fission complex. MFF participates in mitochondrial fission by serving as one of multiple receptors for the GTPase dynamin-related protein 1 (Drp1) (1-4). Research studies have also shown that MFF is a peroxisomal membrane protein and participates in peroxisome fission by serving as a receptor for another GTPase, dynamin-like protein 1 (5,6).
Research studies have demonstrated that the ability of MFF to drive acute mitochondrial fission in response to mitochondrial stress is controlled by AMPK-dependent phosphorylation. AMPK directly phosphorylates MFF at two sites to allow for enhanced recruitment of Drp1 to the mitochondra (7). Multiple isoforms of MFF are expressed as a result of alternative splicing (8). One of the major phosphoacceptor sites of MFF (Ser172 of human isoform 1/Ser146 of human isoforms 2-5) lies within an AMPK phsophorylation motif that spans the boundary of differentially spliced exons of MFF isoforms, suggesting that MFF splice isoforms are phosphorylated by AMPK to varying degrees.
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