Western blot analysis of extracts from various cell lines and tissues using C9orf72 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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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
C9orf72 Antibody recognizes endogenous levels of total C9orf72 protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala202 of human C9orf72 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The expansion of hexanucleotide GGGGCC repeats in the C9orf72 gene causes chromosome 9p-linked neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (1,2). The specific mechanism by which of these repeats contributes to disease etiology is currently an active area of investigation (3). Several gain of function mechanisms have been proposed. These mechanisms include toxicity from C9orf72 RNA containing the hexanucleotide repeats (4) and toxicity generated from dipeptide repeat proteins produced by repeat-associated non-ATG translation (5). In addition to gain of function mechanisms, the genetic hexanucleotide repeat expansions may cause a loss of function of the C9orf72 protein. C9orf72 contains a predicted DENN (differentially expressed in normal and neoplastic cells) domain that typically functions as guanine exchange factors for Rab GTPases, proteins that play key regulatory roles in membrane trafficking (6). Consistent with C9orf72 normally functioning in membrane trafficking, biochemical and genetic studies revealed that C9orf72 forms a protein complex with Sim-Magenis chromosome region 8 (SMCR8) and WD repeat-containing protein 41 (WDR41) to regulate the autophagy-lysosomal pathway (7), suggesting that C9orf72-dependent alterations in the autophagy-lysosomal pathway might contribute to ALS/FTD pathology.
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