Western blot analysis of extracts from L-540 cells, untreated or pre-incubated with either a site-specific phosphorylated peptide to block the signal or a site-specific non-phosphorylated peptide, using Phospho-TNK1 (Tyr277) (D46E7) Rabbit mAb. L-540 cells express a 58 kDa TNK1-C17orf61 fusion protein containing 466 amino acids from the amino terminus of TNK1 [Gu, T.L. et al. (2010) Leukemia].
|MW (kDa)||72 TNK1, 58 TNK1-C17orf61|
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 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
Phospho-TNK1 (Tyr277) (D46E7) Rabbit mAb detects endogenous levels of TNK1 protein only when the amplified gene product is phosphorylated at Tyr277.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr277 of human TNK1 protein.
Tyrosine kinase non-receptor 1 (TNK1) is related to the Ack1 (TNK2) non-receptor kinase that binds cdc42 and inhibits GTPase activity of this cell cycle regulator. TNK1 is broadly expressed in embryogenic tissues and leukemia cell lines, but is restricted to select adult tissues (1). TNK1 is a putative 72 kDa protein comprised of an N-terminal kinase domain, a central SH3 domain and a proline-rich tail. Interaction with PLCγ in vitro indicates a possible role in phospholipid signal transduction pathways (2). Though the exact mechanism is currently unclear, active TNK1 may play a role in regulating cell death by preventing TNF-α-induced NF-κB transactivation (3).
Phosphorylation of TNK1 on Tyr277 was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for phosphorylation site discovery (4) and also reported independently in another publication using MS technology (5). Phosphorylation of TNK1 at Tyr277 was observed in select carcinoma cell lines and in tumors. A constitutively active, 58 kDa truncated TNK1 kinase resulting from fusion between the TNK1 and C17orf61 genes is seen in some cells (5). For additional information, visit PhosphoSitePlus™, CST's modification site knowledgebase, at www.phosphosite.org.
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