Western blot analysis of extracts from various cell lines using Crry Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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.
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
Crry Antibody recognizes endogenous levels of total Crry protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu99 of mouse Crry protein. Antibodies are purified by peptide affinity chromatography.
Crry, also known as complement component receptor 1-like protein (Cr1l), is a murine membrane-bound complement regulatory protein ubiquitously expressed in mouse tissues (1,2). Crry is a functional analog of the human complement decay-accelerating factor (DAF/CD55) and membrane cofactor protein (MCP/CD46) (3). In mice, Crry acts as a cofactor for complement factor I, a serine protease which protects autologous cells against complement-mediated injury by cleaving C3b and C4b deposited on host tissues (4,5). Crry also acts as a decay-accelerating factor, preventing the formation of C4b2a and C3bBb, which are amplification convertases of the complement cascade (3,6). Crry plays a crucial role in early embryonic development by maintaining fetomaternal tolerance (7). On CD4+ T cells, Crry acts as a costimulatory factor which favors IL-4 rather than IFN-gamma secretion, suggesting Th2 polarization (8). Cancer cells and cells of the tumor microenvironment have been shown to over-express Crry, suppressing tumor-specific T cell responses and complement-mediated anti-tumor immunity (9,10). Multiple isoforms have been identified computationally.
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