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.
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: 262
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.
ALPP (3E5C7) Mouse mAb recognizes endogenous levels of total ALPP protein. This antibody may cross-react with a protein of unknown origin at ~140 kDa in some cell lines.
Monoclonal antibody is produced by immunizing animals with a recombinant protein fragment specific to human ALPP protein.
Mammalian alkaline phosphatases (APs) are highly conserved zinc-containing allosteric enzymes that are able to hydrolyze and transphosphorylate a wide range of compounds (1). There are four known human alkaline phosphatase isozymes: TNAP (tissue-nonspecific; bone/liver/kidney), ALPP (placental), ALPP2 (germ cell), and ALPI (intestinal) (2). Placental alkaline phosphatase (ALPP) is bound to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor (3). It is expressed primarily in the placenta (4) and may be involved in transplacental IgG transport (5). ALPP has been found to be overexpressed on the surface of several different types of solid tumor cells (6) and elevated serum concentrations of ALPP and ALPP-like enzymes has been found to be associated with ovarian, cervical, and testicular cancer (7).
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