Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human CXCR4 wild-type protein (CXCR4-WT-Myc/DDK), Myc/DDK-tagged full-length human CXCR4 S324A protein (CXCR4-S324A-Myc/DDK), Myc/DDK-tagged full-length human CXCR4 S325A protein (CXCR4-S325A-Myc/DDK), Myc/DDK-tagged full-length human CXCR4 S324A/S325A protein (CXCR4-S324A/S325A-Myc/DDK), or Myc/DDK-tagged full-length human CXCR4 S339A protein (CXCR4-S339A-Myc/DDK), using Phospho-CXCR4 (Ser324/Ser325) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from MOLT-4 cells, untreated (-) or treated (+) with combinations of the folowing treatments as indicated: CXCL12 (100 nM, 10 min), λ-phosphatase, using Phospho-CXCR4 (Ser324/Ser325) Antibody (upper) and β-actin (D6A8) Rabbit mAb #8457 (lower).
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-CXCR4 (Ser324/Ser325) Antibody recognizes endogenous levels of CXCR4 protein only when phosphorylated at Ser324 and Ser325. This antibody does not cross-react with CXCR4 protein when phosphorylated at Ser339.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Hamster, Chicken
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser324/Ser325 of human CXCR4 protein. Antibodies are purified by protein A and peptide affinity chromatography.
CXCR4 is a chemokine receptor that belongs to the G protein-coupled receptor family. It is activated by a small cytokine, CXCL12, also known as stromal cell derived factor 1 (SDF-1) (1). The main function of CXCR4 is the mediation of the homing of progenitor cells in the bone marrow and their recruitment to sites of injury (2). More recently, CXCR4 has been studied, as a potential therapeutic target, in the context of autoimmune diseases (3) as well as cancer, as the receptor is involved in the regulation of migration, proliferation, and survival of cancer cells (4).
CXCR4 is rapidly phosphorylated at Ser324 and Ser325 by protein kinase C and G protein-coupled receptor kinase 6 upon treatment of cells with CXCL12 (2). Phosphorylation of CXCR4 at Ser324/Ser325 is critical to the ubiquitination and degradation of CXCR4 (6,7).
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