Western blot analysis of extracts from COS-7 cells, untreated or treated with TPA (#4174, 200 nM, 20 min), using the Phospho-(Ser/Thr) PKD Substrates Antibody.
Phospho-(Ser/Thr) PKD Substrates Antibody DELFIA® Assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (T* and S* denote phosphorylated threonine and serine.) (DELFIA® is a registered trademark of PerkinElmer, Inc.)
|Peptide ELISA (DELFIA)||1:1000|
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
(DELFIA® is a registered trademark of PerkinElmer, Inc.)
posted June 2005
revised September 2007
Protocol Id: 34
Phospho-(Ser/Thr) PKD Substrates Antibody detects peptides and proteins containing a phospho-Ser/Thr residue with arginine at the -3 position and leucine at the -5 position, prefering proline at the -1 position within the motif. The antibody may cross-react with some peptides and proteins containing an arginine residue at -3 position and a hydrophobic or basic residue at the -5 position. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)Species Reactivity:
All Species Expected
Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-PKD substrate peptide. Antibodies are purified by protein A and peptide affinity chromatography.
Protein kinase D1 (PKD1), also called PKCμ, is one member of the protein kinase D family. PKD members are serine/threonine protein kinases implicated in very diverse cellular functions including cell growth and survival, Golgi organization and immune response (1). The information about the natural substrates of PKD is limited (2,3). The enzyme recognizes and phosphorylates the following consensus sequence: LXRXXS/T. Phospho-(Ser/Thr) PKD Substrate Antibody recognizes phosphorylated PKD substrates at their consensus motif, providing a powerful new tool for PKD target discovery and characterization as well as HTS drug screening for potential kinase regulators.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.