Western blot analysis of extracts from indicated mouse (wildtype [WT] and FBXO7 knockout [KO]) cerebellum and human cerebellum using FBXO7 (D8L4E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Mouse FBXO7 KO tissue kindly provided by Dr. Judith Stegmüller at the University Hospital RWTH Aachen in Aachen, Germany (3).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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
FBXO7 (D8L4E) Rabbit mAb recognizes endogenous levels of total FBXO7 protein. FBXO7 (D8L4E) Rabbit mAb detects two bands bands in human cerebellum, consistent with multiple FBXO7 isoforms generated by alternative splicing.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the N-terminus of human FBXO7 protein.
F-box only protein 7 (FBXO7) is the substrate recognition component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. This complex mediates ubiquitination and degradation of targeted proteins. Several “PARK” chromosomal loci have been associated with monogeneic forms of PD, including PARK15 (1). The associated allele for PARK15 is FBXO7, which encodes the FBXO7 protein. Mutations in FBXO7 cause early-onset autosomal recessive PD, and alterations in FBXO7 normal function, e.g. the protein degradation process, may contribute to PD etiology (2, 3). Additionally, expression of specific FBXO7 isoforms generated by alternative splicing is linked with PD (4).
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