Western blot analysis of extracts from B16-F10 cells, murine bone marrow-derived macrophages (mBMDMs), and EL4 cells using GPNMB Antibody (Mouse Specific) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, GPNMB protein is not expressed in EL4 cells.
Western blot analysis of extracts from B16-F10 cells, mock transfected (-) or transfected with siRNA targeting mouse GPNMB (+), using GPNMB Antibody (Mouse Specific) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from B16-F10 cells, untreated (-) or treated with PNGase F (+), using GPNMB Antibody (Mouse Specific) (upper) and 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 mouse GPNMB protein (mGPNMB-Myc/DDK; +) and Myc/DDK-tagged full-length human GPNMB protein (hGPNMB-Myc/DDK; +), using GPNMB Antibody (Mouse Specific) (upper), DYKDDDDK Tag Antibody #2368 (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 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
GPNMB Antibody (Mouse Specific) recognizes endogenous levels of total mouse GPNMB protein. This antibody does not cross-react with human GPNMB protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of mouse GPNMB protein. Antibodies are purified by protein A and peptide affinity chromatography.
Glycoprotein non-metastatic gene B (GPNMB) is a type I transmembrane glycoprotein over expressed in many types of cancer. The GPNMB glycoprotein is involved in many physiological processes, including mediating transport of late melanosomes to keratinocytes (1), regulating osteoblast and osteoclast differentiation and function (2), stimulating dendritic cell maturation, promoting adhesion of dendritic cells to endothelial cells (3), enhancing autophagosome fusion to lysosomes in tissue repair, and regulating degradation of cellular debris (4,5).
While typical GPNMB expression is seen in tissues including skin, heart, kidney, lung, liver, and skeletal muscle (3,6), research studies show elevated GPNMB expression often contributes to the metastatic phenotype in numerous cancers (reviewed in 7). GPNMB is typically localized to intracellular compartments in normal cells (1,8), but investigators found it primarily on the cell surface of tumor cells (9,10). Differential localization and expression, and the role of GPNMB as a tumor promoter in many cancer types make this protein a viable therapeutic target (11).
The GPNMB ectodomain can be cleaved by matrix metalloproteinases and shed from the cell surface (12). Research studies identify the sheddase ADAM10 as one peptidase responsible for cleavage of the GPNMB ectodomain at the surface of breast cancer cells. Shedded GPNMB ectodomains may promote angiogenesis by inducing endothelial cell migration (13).
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