|H M R||Endogenous||35||Rabbit IgG|
Western blot analysis of extracts from various cell lines using Pim-3 (D17C9) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of recombinant Pim-1, 2, and 3 using Pim-3 (D17C9) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from COS-7 cells, untransfected () or transfected with a mouse Pim-3 construct (+), using Pim-3 (D17C9) Rabbit mAb.Learn more about how we get our images.
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, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Pim-3 (D17C9) Rabbit mAb detects endogenous levels of total Pim-3 protein. It does not cross-react with other Pim family members.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser275 of human Pim-3.
Pim proteins (Pim-1, Pim-2 and Pim-3) are oncogene-encoded serine/threonine kinases (1). Pim-1, a serine/threonine kinase highly expressed in hematopoietic cells, plays a critical role in the transduction of mitogenic signals and is rapidly induced by a variety of growth factors and cytokines (1-4). Pim-1 cooperates with c-Myc in lymphoid cell transformation and protects cells from growth factor withdrawal and genotoxic stress-induced apoptosis (5,6). Pim-1 also enhances the transcriptional activity of c-Myb through direct phosphorylation within the c-Myb DNA binding domain as well as phosphorylation of the transcriptional coactivator p100 (7,8). Hypermutations of the Pim-1 gene are found in B-cell diffuse large cell lymphomas (9). Phosphorylation of Pim-1 at Tyr218 by Etk occurs following IL-6 stimulation and correlates with an increase in Pim-1 activity (10). Various Pim substrates have been identified; Bad is phosphorylated by both Pim-1 and Pim-2 at Ser112 and this phosphorylation reverses Bad-induced cell apoptosis (11,12).
Pim-3, originally named Kid-1, was identified as a protein induced by depolarization in PC-12 cells (13). Like other family members, Pim-3 can phosphorylate Bad to inhibit Bad-mediated apoptosis (14). Aberrant expression of Pim-3 has been observed in a number of types of cancer, including liver, pancreas, colon and gastric cancer (14-17).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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