REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H Mk | Endogenous | 120 | Rabbit IgG |
Western blot analysis of exracts from LN18, HT-29, and U-2 OS cells using α-Adducin (D7T7R) Rabbit mAb.
Learn more about how we get our imagesImmunoprecipitation of α-adducin from U-2 OS cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is α-Adducin (D7T7R) Rabbit mAb. Western blot analysis was performed using α-Adducin (D7T7R) Rabbit mAb.
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 utilizing magnetic separation.
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.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
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) (D4W3E) mAb (#45262) 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 October 2017
Protocol Id: 410
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
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.
α-Adducin (D7T7R) Rabbit mAb recognizes endogenous levels of total α-adducin protein.
Human, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp643 of human α-adducin protein.
The adducins (ADD) are cytoskeleton-associated proteins that help cap the ends of actin filaments, promote association between spectrin and actin, and participate in synapse assembly. The three closely related genes ADD1, ADD2, and ADD3 encode the α-adducin, β-adducin, and γ-adducin proteins (1). Research studies indicate that β-adducin is found at high levels in brain and hematopoietic tissues, whereas both α-adducin and γ-adducin are ubiquitously expressed (2). Adducin protein function is regulated by phosphorylation at a number of sites. Both PKA and PKC can phosphorylate α-adducin at Ser726 and β-adducin at Ser713, which inhibits calmodulin binding and adducin activity (3-5). Additionally, PKA (but not PKC) can phosphorylate β-adducin at Ser408, Ser436, and Ser481, which negatively affects spectrin-actin interactions (3). Phosphorylation of α-adducin at Thr445 and Thr480 by Rho-kinase regulates cell motility and membrane ruffling (6). Finally, CDK-1 phosphorylation of α-adducin at Ser12 and Ser355 during mitosis leads to association of α-adducin with the mitotic spindle, suggesting that α-adducin may play a role in mitotic regulation (7). Because α-adducin plays a role in regulating renal sodium reabsorption, it is not surprising that a number of studies show a relationship between ADD1 genetic polymorphisms and the development of hypertension (8-10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.
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Product # | Size | Price |
---|---|---|
70174S | 100 µl (10 western blots) | In Cart |
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