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CXCR4 / CD184 Antibody (PA3-305) in WB
Western blot analysis of CXCR4 was performed by loading 25ug of Ramos, Raji, and Jurkat whole cell lysates or membrane protein fractions, generated using IP Lysis Buffer (Product ## 87787) and Mem-PER Plus Membrane Protein Extraction Kit (Product ## 89842), respectively, and 10ul of PageRuler Prestained Protein Ladder (Product ## 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Blotter (Product ## 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. CXCR4 was detected at ~60kD and 72kD using CXCR4 polyclonal antiserum at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product ## 31460) at a dilution of 1:40,000 for at least 30 minutes. Chemiluminescent detection was performed using SuperSignal West Dura (Product ## 34076). NOTE: The presence of two distinct bands with this antibody likely represents the detection of two different isoforms and/or post-translationally modified forms of CXCR4, many of which have been described in the literature. For optimal detection of CXCR4 by Western blot, it is recommended to first use a membrane extraction buffer for efficient protein isolation.
