Western blot analysis of extracts from various cell lines using PINCH (5G7) Mouse mAb.
|REACTIVITY||H M R Mk|
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.
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.
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 June 2016
Protocol Id: 19
PINCH (5G7) Mouse mAb recognizes endogenous levels of total PINCH protein. The antibody recognizes a protein of unknown origin at 80 kDa in some cell lines.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to the the human PINCH protein.
The extracellular matrix (ECM) is a complex structure of secreted macromolecules surrounding mammalian organs and tissues. Controlled interactions between cells and the ECM are important in proliferation, migration, survival, polarity, and differentiation. Cells contact the ECM primarily through focal adhesion complexes, which contain integrins, as well as multiple adaptor and signaling proteins (1). The ILK/PINCH/Parvin (IPP) adaptor complex acts at the interface of the integrin/actin connection to regulate formation of focal adhesions and integrin signaling. Roles for IPP proteins outside of the IPP complex have been proposed, including regulation of gene expression (2,3).
PINCH, also known as LIMS1, has been shown to function as a specific regulator of gene expression in glomerular podocytes in response to TGF-β1 (4). Researchers have shown that PINCH is highly expressed in some human tumors, and that PINCH can promote resistance to ionizing radiation through activation of Akt (5,6).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.