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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
HHLA2/B7-H7 (E1U6X) Rabbit mAb recognizes endogenous levels of total HHLA2/B7-H7 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asn101 of human HHLA2/B7-H7 protein.
HERV-H LTR-associating protein 2 (HHLA2, with alternative names of B7-H5 and B7-H7) is a member of the B7 immunoglobulin superfamily (1). HHLA2 protein is constitutively expressed on the surface of human monocytes and is induced on B cells after stimulation with LPS and IFN-γ (1,2). Through interaction with TMIGD2, which is constitutively expressed on all naïve T cells and the majority of natural killer cells, but not on T regulatory cells or B cells, HHLA2 co-stimulates T cells in the context of TCR-mediated activation, enhancing T cell proliferation and cytokine production via an AKT-dependent signaling cascade (2). Contrary to this, HHLA2 has also been shown to inhibit T cell proliferation and cytokine production, suggesting a secondary receptor for HHLA2 that is expressed on activated T cells with co-inhibitory functions (3). Moreover, HHLA2 has been shown to be highly expressed in various types of cancer, and is associated with a poor prognosis (4-10). Further understanding the immunologic functions of the HHLA2 pathway will guide the selection of agents used for cancer immunotherapy, autoimmune disorders, infection, and transplant rejection.
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