Western blot analysis of extracts from KM-12 and OVCAR-3 cells using REG4 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, OVCAR-3 are low or negative for REG4.Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
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.
REG4 Antibody recognizes endogenous levels of total REG4 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human REG4 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Reg4 (regenerating islet-derived protein 4) is a member of a multigene family of Reg proteins (1). Reg proteins are secreted glycoproteins belonging to the calcium (C-type) dependent lectin superfamily, although they can in fact bind polysaccharides, mannan, and heparin in the absence of calcium (2,3).
Reg4 is expressed in the gastrointestinal (GI) tract, in normal colon mucosa, and is up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, inflammatory bowel disease (Crohn’s disease and ulcerative colitis) (4,5).
It has recently been shown that Reg4 marks a population of deep secretory cells at the bottom of the crypts in the colon. In the upper gastrointestinal tract, Paneth cells support the Lgr5+ positive stem cells that reside at the base of deep crypts by providing signals such as Wnt3, EGF, and Notch ligands for stem cell maintenance. No Paneth cells however exist in the colon crypts. It is proposed that the Reg4+ population serves as an analogous support system for the LGR5+ positive stem cell population in the colon (1,6).
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|43411S||100 µl (10 western blots)||$ 255.0|