Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human GLTSCR2 protein (hGLTSCR2-Myc/DDK; +), using GLTSCR2 Antibody.
Western blot analysis of extracts from various cell lines using GLTSCR2 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
GLTSCR2 Antibody recognizes endogenous levels of total GLTSCR2 protein. .Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys410 of human GLTSCR2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2), also known as protein interacting with carboxyl terminus-1 (PICT-1), displays tumor suppressor activity, in part, by interacting with the C-terminal region of PTEN and preventing its degradation. Disruption of this interaction upregulates activity of the PI3K/Akt signaling axis and promotes cell transformation (1,2). Further evidence to support the tumor suppressor role of GLTSCR2 lies in the finding that GLTSCR2 is localized to the tumor suppressive region of chromosome 19q, which is subject to lesions in human brain tumors (3). Indeed, research studies have demonstrated that GLTSCR2 harbors nonsense mutations and deletions in glioblastomas, which lead to a decrease in GLTSCR2 protein expression (4). GLTSCR2 has also been shown to exert tumor suppressor activity through its involvement in the nucleolar stress response. Research studies indicate that in response to ribosomal stress, GLTSCR2 translocates from the nucleolus to the nucleoplasm where it binds and stabilizes p53 tumor suppressor, which results in inhibition of cell cycle progression (5).
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