Western blot analysis of extracts from HaCaT cells, untreated (-) or treated with Human Transforming Growth Factor β1 (hTGF-β1) #8915 (10 ng/mL, 18 hr; +), using p15 INK4B (E3R6S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
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
p15 INK4B (E3R6S) Rabbit mAb recognizes endogenous levels of total p15 INK4B protein. This antibody does not cross-react with p16 INK4A.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg133 of human p15 INK4B protein.
Members of the INK4 family of cyclin dependent kinase inhibitors include p16 INK4A, p15 INK4B, p18 INK4C, and p19 INK4D. The INK4 family members inhibit cyclin dependent kinases 4 and 6 (CDK4 and CDK6), causing cell cycle arrest in G1 phase. The INK4A-ARF-INK4B locus on chromosome 9p21, frequently lost in human cancer, encodes the INK4 family members p16 INK5A and p15 INK4B, as well as the unrelated protein, ARF (1).
p15 INK4B protein expression is induced by TGF-β in human keratinocytes (2), a process which requires demethylation at the p15 INK4B locus (3). In hematopoietic cells, p15 INK4B signaling functions in the determination of cell fate (4,5).
Researchers have shown that expression levels of p15 INK4B, altered by epigenetic modification, have an impact on disease progression in multiple myeloma (6). Similarly, studies in pancreatic cancer have shown hypermethylation and epigenetic silencing of p15 INK4B, p16 INK4A, and CDK inhibitors p21cip1 and p27kip1 (7).
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