Western blot analysis of extracts from HCT 116 cells, G0 cells that were serum starved and contact inhibited (36 hr), or mitotic cells treated with thymidine (2 mM, 18 hr) followed by Nocodazole #2190 (50 ng/ml, 20 hr), using Phospho-MAPK Substrates Motif [PXpTP] MultiMab™ Rabbit mAb mix (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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-MAPK Substrates Motif [PXpTP] MultiMab™ Rabbit mAb mix recognizes endogenous levels of proteins that are phosphorylated at threonine within the context of a PXTP motif. The antibody does not cross-react with endogenous levels of non-phosphorylated proteins, phospho-threonine in another context, or proteins with a phosphorylated serine within the PXSP motif.Species Reactivity:
All Species Expected
MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. Each antibody in the mix is carefully selected based on motif recognition and performance in multiple assays. Each mix is engineered to yield the broadest possible coverage of the modification being studied while ensuring a high degree of specificity for the modification or motif.
The MAPK and CDK families of serine/threonine protein kinases play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (1-3). MAPK phosphorylates substrates with the consensus sequence PX(S/T)P, and CDKs phosphorylate substrates containing the consensus sequence (S/T)PXR/K. Cell Signaling Technology has developed antibodies that bind to phospho-threonine followed by proline, motifs PXS*/T*P and/or S*PXR/K, for use in the study and discovery of new MAPK and CDK substrates (4,5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MultiMab is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, 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.