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
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.
TIAM1 Antibody recognizes endogenous levels of total TIAM1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human TIAM1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
TIAM1 (T-lymphoma invasion and metastasis-inducing protein 1) is a multidomain guanine nucleotide exchange factor (GEF) protein that activates Rac1, a GTPase involved in cytoskeletal dynamics that regulate cell migration, growth and survival. TIAM1 also has been identified as an inhibitor of the YAP/TAZ signaling pathway, with two distinct subcellular mechanisms of action: (1) promoting cytoplasmic (proteosomal) degradation of YAP and TAZ; and (2) blocking the transcriptional co-activator functions of YAP and TAZ in the nucleus (3,4). The effects of TIAM1 on tumor development are also complex and context-dependent. For example, it has been reported that TIAM1 can promote tumor growth and progression in some contexts, while antagonizing tumor metastasis and invasion in other contexts (5,6).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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