Western blot analysis of extracts from various cell lines using Nischarin (D6T4X) rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with full-length human Nischarin-Myc/DDK (hNischarin-Myc/DDK; +), using Nischarin (D6T4X) rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
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
Nischarin (D6T4X) Rabbit mAb recognizes endogenous levels of total nischarin protein. The antibody detects a 60 kDa background band of unknown identity by western blot.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human nischarin protein.
Nischarin (also known as imidazoline receptor antisera-selected protein, IRAS) was first identified through its association with the cytoplasmic domain of integrin α5, and shown to regulate cell migration and cystoskeletal organization (1).
Nischarin regulates Rac-1 signaling (2), as well as the p21-activated kinase (PAK) (3) and cofilin/LIMK pathways (4). Nischarin also interacts with LKB1, regulating the migration and metastatic behavior of breast epithelial cells (5). In addition, nischarin regulates neuronal migration in rat brain (6).
Research studies have implicated nischarin in the regulation of invasion and metastasis of breast cancer (7,8). Researchers have shown that nischarin is frequently downregulated in ovarian cancer, and regulates invasion through focal adhesion kinase (FAK) signaling (9).
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