Western blot analysis of extracts from various cell lines using PAPSS2 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). No signal is detected in SF-539 cells, which contain a homozygous null mutation at the PAPSS2 locus, confirming specificity of the antibody.
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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
PAPSS2 Antibody recognizes endogenous levels of total PAPSS2 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro567 of human PAPSS2 protein. Antibodies are purified by peptide affinity chromatography.
PAPSS2 is a bifunctional enzyme with both ATP sulfurylase and APS kinase activity, which mediates two reactions to generate 3'-Phosphoadenosine-5'-phosphosulfate (PAPS), a critical intermediate in the sulfate activation pathway. PAPS is the sulfate donor co-substrate for all sulfotransferase (SULT) enzymes and is required to catalyze the sulfate conjugation of many endogenous and exogenous compounds (1,2). Mutations in PAPSS2 lead to an autosomal recessive form of spondyloepimetaphyseal dysplasia (SEMD) in humans (3), whereas mutant mice lacking PAPSS2 activity demonstrate defective postnatal skeletal development leading to premature joint degeneration and brachymorphism (4). In conjunction with SULT1E1, PAPSS2 and its paralogue PAPSS1 are responsible for sulfation and subsequent inactivation of estrogen in target tissues. Expression levels of PAPSS2 were found to be significantly higher in tumorous breast and endometrial tissues than in adjacent normal tissues, suggesting that targeting PAPSS2 could be an important approach for estrogen-dependent cancers (5). Additionally, PAPSS2 provides the universal sulfate donor PAPS to SULT2A1, which is responsible for sulfation of the crucial androgen precursor dehydroepiandrosterone (DHEA) (6). TGF-β signaling has been shown to regulate the expression of PAPSS2 via stabilization of SOX9 protein in mouse articular cartilage and bovine chondrocytes (7,8).
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