Western blot analysis of extracts of 293 and HeLa cells, either untreated or treated with UV radiation, using Phospho-PNK1 (Ser114/Thr118) Antibody (upper). β-actin Antibody #4967 (lower) was used to control for loading.
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
Phospho-PNK1 (Ser114/Thr118) Antibody detects endogenous levels of PNK1 only when phosphorylated on Ser114 and Thr118.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser114 and Thr118 of human PNK1. Antibodies are purified by protein A and peptide affinity chromatography.
PNK (polynucleotide kinase) is a DNA repair enzyme that participates in single strand break repair and non-homologous end rejoining (NHEJ) for double strand breaks. PNK possesses a 5'-DNA kinase activity and a 3'-DNA phosphatase activity (1,2). It has three domains, a C-terminal kinase domain, a central phosphatase domain, and an N-terminal forkhead associated (FHA) domain that is responsible for protein-protein interactions. Reduction in expression of PNK by RNAi sensitizes cells to ionizing radiation and topoisomerase I inhibitors (3)
Phosphorylation of PNK1 at Ser114/ Thr118 was independently identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's MS/MS platform for phosphorylation site discovery. Phosphorylation of PNK1 at Ser114/ Thr118 was observed in extracts of various cell lines following UV treatment. For additional information please visit PhosphoSitePlus®, CST's modification site knowledgebase, at www.phosphosite.org.
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