|H M R Mk||Endogenous||15||Rabbit|
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 and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
PHLDA3 Antibody detects endogenous level of total PHLDA3 protein.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human PHLDA3 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Pleckstrin homology-like domain family A member 3 (PHLDA3) is one of three relatively small, similar proteins that share a common pleckstrin homology (PH) domain. Expression of PHLDA3 and the related PHLDA1 occurs in several fetal and adult tissues, in contrast to the more restricted expression of PHLDA2 seen in mouse tissues (1). PHLDA3 is one of several proteins induced following treatment of tumor cells with cisplatin, an anti-cancer drug that cross-links DNA and promotes apoptosis through activation of the tumor suppressor p53 (2). Additional evidence that PHLDA3 is involved in promoting apoptosis through p53 came from a study examining the opposing effects of p53 and Akt in tumor development. Tumor suppressor p53 binds the PHLDA3 promoter to induce transcription. Induced overexpression of PHLDA3 increases apoptosis while deletion of PHLDA3 results in increased Akt activity and a reduction in p53-mediated apoptosis. PHLDA3 appears to compete with the PH domain of Akt, preventing Akt activation and promotion of Akt-induced cell survival pathways (3).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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