Western blot analysis of extracts from Ramos and A20 cells, untreated (-), or treated (+) with either anti-human IgM (12 μg/ml, 10 min), or H2O2 (11 mM, 1 min), using Phospho-BLNK (Thr152) Antibody (upper), BLNK (D3P2H) XP® Rabbit mAb #36438 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from Ramos cells, untreated (-) or treated with anti-human IgM (12 μg/ml, 10 min; +), and lambda-phosphatase and calf intestinal phosphatase (λ-phosphatase/CIP; +) as indicated, using Phospho-BLNK (Thr152) Antibody (upper), BLNK (D3P2H) XP® Rabbit mAb #36438 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
|MW (kDa)||68, 70|
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.
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
Phospho-BLNK (Thr152) Antibody recognizes endogenous levels of BLNK protein only when phosphorylated at Thr152.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr152 of human BLNK protein. Antibodies are purified by protein A and peptide affinity chromatography.
B cell linker protein (BLNK), also known as SLP-65 or BASH, is an adaptor molecule that plays key roles in B cell activation and B cell antigen receptor (BCR) engagement. BLNK acts at the interface between BCR-associated Syk and downstream signaling cascades (1,2). BLNK has multiple SH2 binding motifs (YXXP) at its amino terminus and an SH2 domain at its carboxy terminus. After BCR ligation, BLNK is phosphorylated by Syk at multiple YXXP motifs including Tyr72, Tyr84, Tyr96, and Tyr178 (1). These phosphorylated motifs provide docking sites for signaling molecules, such as BTK, PLCγ, and Vav. These signaling molecules bind to BLNK through their SH2 domains and together activate downstream signaling pathways (3,4). Through its SH2 domain, BLNK can also interact with tyrosine-phosphorylated targets, such as HPK1, thereby recruiting them to the BCR complex for signaling (5).
Following BCR ligation, BLNK is phosphorylated at Thr152 by the hematopoietic progenitor kinase 1 (HPK1) (6). This phosphorylation induces interaction with 14-3-3ε, which leads to the disassembly of BCR signaling complexes and downregulation of BCR signaling (6).
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