REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H All | Endogenous | Rabbit IgG |
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-CDK Substrate [pTPXK] (D9V5N) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Learn more about how we get our imagesImmunoprecipitation of phospho-TPXK motif-containing proteins from mitotic HCT 116 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 or Phospho-CDK Substrate [pTPXK] (D9V5N) Rabbit mAb. Lane 1 is 10% input. Western blot analysis was performed using Phospho-CDK Substrate [pTPXK] (D9V5N) Rabbit mAb followed by Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 as secondary antibodies.
Learn more about how we get our imagesFor 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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised November 2013
Protocol Id: 409
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
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.
Phospho-CDK Substrate [pTPXK] (D9V5N) Rabbit mAb recognizes endogenous levels of proteins that are phosphorylated at threonine within the context of a TPXK motif. The antibody does not cross-react with non-phosphorylated proteins or phospho-threonine in a different context.
Human, All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide library containing the phospho-TPXK motif.
Cyclin-dependent kinases (CDKs) are a family of Ser/Thr kinases that regulate cell-cycle transition through their association and subsequent phosphorylation of substrate proteins in a proline-directed fashion. CDKs phosphorylate a serine or threonine residue within the (S/T)PX(R/K) consensus sequence (1-5). Cell Signaling Technology has developed antibodies to phosphorylated proline-rich motifs for broad-based discovery applications. The TPXK motif is targeted for phosphorylation by CDKs 1, 2, 4, 5, and 6 (1-5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PTMScan 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 ptmscan@cellsignal.com.
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Product # | Size | Price |
---|---|---|
14371S | 100 µl (10 western blots) | $ 297.0 |