Western blot analysis of extracts from NK-92 cells, untreated (-) or treated as indicated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 5 hr; +) and Ionomycin, Calcium Salt #9995 (3 μM, 5 hr; +), or Brefeldin A #9972 (300 ng/mL, last 4 hr of stimulation; +), using XCL1/XCL2 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human XCL1 protein (hXCL1-Myc/DDK; +) and Myc/DDK-tagged full-length human XCL2 protein (hXCL2-Myc/DDK; +), using XCL1/XCL2 Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), 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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
XCL1/XCL2 Antibody recognizes endogenous levels of total XCL1 and XCL2 proteins.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro99 of human XCL1 protein. Antibodies are purified by peptide affinity chromatography.
XCL1 (Lymphotactin, SCM-1 alpha, Chemokine (C motif) ligand 1) and XCL2 (SCM-1 beta, Chemokine (C motif) ligand 2) comprise the C chemokine family, lacking two of the four invariant cysteines normally found on chemokines (1,4). Their sequences differ by 2 amino acids and they are primarily expressed by CD8+ T cells and NK cells (1-5). XCL1 and XCL2 act as ligands for the chemokine receptor XCR1 (1-5). XCR1-positive dendritic cells cross-present antigens to naïve CD8+ T cells, priming them to become activated cytotoxic CD8+ T cells (6-7). In addition, in mouse models, the XCL1-XCR1 signaling axis was shown to be involved in the formation of self-tolerance through the development of Treg cells within the thymus (8).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.