Western blot analysis of extracts from mouse testis and mouse spleen using Ly6k Antibody (Mouse Specific) (upper) and β-actin #8457 (lower). Mouse spleen is negative or low for Ly6k expression as expected.
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
Ly6k Antibody (Mouse Specific) recognizes endogenous levels of total Ly6k protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro70 of murine Ly6k protein. Antibodies are purified by protein A and peptide affinity chromatography.
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).
Ly6k is a member of the Ly6/urokinase type plasminogen activator receptor-like protein (uPAR)(LU) superfamily. It is expressed in testes where in interaction with another (uPAR)(LU) family member, TEX101, it is proposed to be necessary for the production of viable sperm (4). The human ortholog of this protein, LY6K, has been characterized to be a cancer testes antigen. It is found to be upregulated in numerous types of cancer, including esophageal squamous cell carcinoma, bladder cancer, and breast cancer (5,6).
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