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
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.
PEAK1 (D4G6J) Rabbit mAb recognizes endogenous levels of total PEAK1 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr695 of human PEAK1 protein.
PEAK1 (Pseudopodium-enriched atypical kinase 1 or sgk269) is a member of nonreceptor atypical tyrosine kinase family identified by MS analysis of purified psedopodium (1). PEAK1 is a multi-domain protein with a N-terminal Erk binding site, followed by actin-targeting/Src substrate/Erk substrate region, Crk binding site, Shc binding site, and a C-terminal kinase domain (1, 2). By interacting with different adaptors like Shc, Grb2, Src, and others, PEAK1 functions as an important regulator in different signaling pathways, namely the Src/PEAK1/ErbB2 (3), EGFR Shc1/PEAK1/Grb2(4), TGFβ/PEAK1/Src/MAPK (5), and fibronectin/PEAK1/Src (6) pathways. PEAK1 plays an instrumental role in a wide variety of biological processes including epithelial-mesenchymal transition (EMT), dynamics of focal adhesion, cancer metastatic growth and invasion as well as cancer drug resistance (3, 5-8). Phosphorylation of PEAK1 at Tyr665 or Tyr635 by SFK (Src family Kinases) has been shown to be essential for cancer cell migration and invasion as well as the turnover of focal adhesions (7, 9).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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