Western blot analysis of extracts from various cell lines using FMRP (G468) Antibody.
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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
FMRP (G468) Antibody detects endogenous levels of total FMRP protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence around Gly468 of human FMRP protein. Antibodies are purified by protein A and peptide affinity chromatography.
Fragile X syndrome, a frequent cause of inherited mental retardation, often results from expansion of the CGG trinucleotide repeat in the gene that encodes the fragile X mental retardation protein (FMRP) (1). FMRP (also known as FMR1) and its two autosomal homologs (FXR1 and FXR2) all bind RNA and play a role in the pathogenesis of fragile X syndrome (1-3). Each of these related proteins can associate with one another as well as form homodimers (3). FMRP can act as a translation regulator and is a component of RNAi effector complexes (RISC), suggesting a role in gene silencing (4). In Drosophila, dFMRP associates with Argonaute 2 (Ago2) and Dicer and coimmunoprecipitates with miRNA and siRNA. These results suggest that fragile X syndrome is related to abnormal translation caused by a defect in RNAi-related pathways (5). In addition, FMRP, FXR1, and FXR2 are components of stress granules (SG) and have been implicated in the translational regulation of mRNAs (6).
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