Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human FAT10 protein (hFAT10-Myc/DDK; +), using FAT10 (D1Q3Y) Rabbit mAb (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
Western blot analysis of extracts from SW620 and Huh7 cells, untreated (-) or treated (+) with combinations of the following treatments as indicated: hIFNγ (100ng/mL, 16 hr) and hTNFα #8902 (100ng/mL, 16 hr), using FAT10 (D1Q3Y) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
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
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.
FAT10 (D1Q3Y) Rabbit mAb recognizes endogenous levels of total FAT10 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human FAT10 protein.
HLA-F adjacent transcript 10 (FAT10/Ubiquitin D) belongs to the ubiquitin-like modifier (Ubl) family of proteins. The 18 kDa FAT10 protein contains two tandem Ubl domains that are oriented in a head-to-tail fashion and a free C-terminal di-glycine motif, which is available for isopeptide bond formation with target proteins via an E1-E2-E3 enzymatic cascade (1). Indeed, FAT10 provides a ubiquitin-independent signal for proteasomal degradation (2). Research studies have demonstrated that FAT10 expression is enriched in lymphoid organs and that its expression is transiently upregulated via the NF-kB pathway in response to pro-inflammatory cytokines such as TNFα and IFNγ (1,3-5). In solid tumors that possess inflammatory microenviroments, research studies have shown that FAT10 is overexpressed and may serve as a biomarker for inflamed tumors (6,7).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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