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, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
NEK7 (C34C3) Rabbit mAb detects endogenous levels of total NEK7 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of human NEK7.
The NEK family of protein kinases is composed of 11 members in humans that share an amino-terminal catalytic domain related to NIMA, a serine/threonine kinase identified in Aspergillus nidulans. While NIMA is critical for cell cycle progression in fungus, the function of NEK kinases in mammalian cells is largely unknown. NEK1 was first identified by screening mouse cDNA expression libraries and was demonstrated to have dual specificity kinase activity on both tyrosine and serine/threonine sites (1). NEK2 most closely resembles fungal NIMA in its primary structure and is believed to promote the splitting of duplicated centrosomes at the onset of mitosis (2,3). NEK3 is predominantly a cytoplasmic enzyme and its activity shows marginal variation throughout the cell cycle (4). NEK4 is ubiquitously expressed and its expression and subcellular location are not associated with cell cycle (5). NEK6/7 have been suggested to phosphorylate and activate p70 S6 kinase in vitro (6). Expression of an inactive NEK6 mutant arrests cells in M phase and interferes with chromosome segregation (7). NEK8 activity is not cell cycle regulated and may play a role in cell cycle independent microtubule dynamics (8). NEK9 is activated during mitosis and may participate in the activation of NEK6/7 during mitosis (9,10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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