Western blot analysis of extracts from HepG2 and HCT116 cells using Mig6 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
Mig6 Antibody detects endogenous levels of total Mig6 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val195 of human Mig6. Antibodies are purified by protein A and peptide affinity chromatography.
Mig6 was identified as a gene which is induced when quiescent fibroblasts are treated by mitogens (1). During cell cycle progression, Mig6 expression levels are also regulated (1). Mig6 mRNA levels were found to increase upon stimulation by chronic stresses including diabetic nephropathy (2). Overexpression of this gene leads to the activation of stress-activated protein kinases (SAPKs)/c-Jun amino-terminal kinases (JNKs) (2). Furthermore, Mig6 was found to interact with epidermal growth factor receptor (EGFR) when stimulated by epidermal growth factor (EGF) (3). Deletion of the Mig6 gene in mice results in hyperactivation of EGFR and signaling through the mitogen-activated protein kinase (MAPK) pathway, causing overproliferation and abnormal differentiation of epidermal keratinocytes in these animals. Inhibition of endogenous EGFR signaling by Iressa abolished skin defects observed in Mig6(-/-) mice, indicating that Mig6 is a specfic negative regulator of EGFR signaling (4). Furthermore, expression of Mig6 was significantly lower in skin, breast, pancreatic and ovarian cancers, suggesting a role of Mig6 as a tumor suppressor (4).
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