Western blot analysis of extracts from HT-1080, HT-29 and COLO-201 cell lines using Maspin (L250) 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 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
Maspin (Q260) Antibody detects endogenous levels of total maspin protein.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu250 of human maspin. Antibodies were purified by peptide affinity chromatography.
Maspin (SERPINB5) was discovered as a mammary tumor suppressor that is expressed in normal mammary epithelium but lost in most breast cancer cell lines (1). While maspin is related to the serpin family of serine protease inhibitors, it may not function as a protease inhibitor (2). It plays an essential role in embryonic development through critical roles in cell adhesion (3). While the precise mechanism of maspin signaling is unclear (4), the tumor suppressing activity of maspin has been attributed to its ability to inhibit cell invasion/metastasis (5,6) and angiogenesis (7), while promoting apoptosis (8). Nuclear translocation of active IKKα has been shown to repress maspin transcription and promote prostate cancer metastasis (9).
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