Western blot analysis of extracts from MCF7 and SK-MEL-28 cells using CDK4 (D9G3E) Rabbit mAb (Biotinylated).
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated CDK4 (D9G3E) Rabbit mAb #12790.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/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.
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* 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 June 2016
Protocol Id: 266
CDK4 (D9G3E) Rabbit mAb (Biotinylated) recognizes endogenous levels of total CDK4 protein. In COS-7 cells, this antibody recognizes a 40 kDa background band of unknown origin. This antibody does not cross-react with CDK1, 2, or 6.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CDK4 protein.
Cyclin-dependent kinase activity is regulated by T-loop phosphorylation (Thr172 in the case of CDK4), by the abundance of their cyclin partners, and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of CDK4/cyclin D and p27 Kip1/Cip1 requires extracellular mitogenic stimuli for the release and degradation of p27, which affects progression through the restriction point and pRb-dependent entry into S-phase (2). The active complex of CDK4/cyclin D targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). In HeLa cells, upon UV irradiation, upregulation of p16 INK4A association with CDK4/cyclin D3 leads to a G2 delay, implicating CDK4/cyclin D3 activity in progression through the G2-phase of the cell cycle (4).
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