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 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 June 2020
Protocol Id: 10
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro93 of human CT45-1 protein.
Cancer/testis antigens (CTAs) are a family of more than 100 proteins whose normal expression is largely restricted to immune privileged germ cells of the testis, ovary, and trophoblast cells of the placenta. Although most normal somatic tissues are void of CTA expression, due to epigenetic silencing of gene expression, their expression is upregulated in a wide variety of human solid and liquid tumors (1,2). As such, CTAs have garnered much attention as attractive targets for a variety of immunotherapy-based approaches to selectively attack tumors (3).
The CT45 family is a tandem cluster of highly similar genes which share many features with other classic CT genes including Xq localization, multigene family, and identical or near-identical gene copies indicating recent gene duplications (4). The nuclear protein antigen CT45 is often highly expressed in lung cancer and ovarian cancer, and the frequency and characteristics of CT45 expression are similar to that of other CT cancer vaccine targets currently in clinical trials, including NY-ESO-1 and MAGE-A (5). Overexpression of CT45A1 in breast cancer cells was found to upregulate various oncogenic and metastatic genes, promote epithelial-mesenchymal transition, and increase cell stemness, tumorigenesis, and metastasis (6). Additionally, research studies have shown that CT45A1 silencing suppresses cell proliferation and metastasis in breast and lung cancer cells by downregulating the ERK/CREB signaling pathway (6,7). Therefore, CT45A1 and other CT45 family members are excellent targets for anticancer drug discovery and targeted tumor therapy.
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