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.
GFAT2 (D40C7) Rabbit mAb recognizes endogenous levels of total GFAT2 protein. This antibody also cross-reacts with GFAT1 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu205 of human GFAT2 protein.
GFAT1, glutamine:fructose-6-phosphate aminotransferase 1, is the rate-limiting enzyme of the hexosamine biosynthesis pathway (1). This enzyme catalyzes the conversion of fructose-6-phosphate and glutamine to glucosamine-6-phosphate and glutamate (2). The hexosamine biosynthesis pathway generates the building blocks for protein and lipid glycosylation (2). Furthermore, studies suggest that increased activity of this pathway is a contributing factor to hyperglycemia-induced insulin resistance (1,2). GFAT1 is more active in non-insulin-dependent diabetes mellitus (NIDDM) patients (3). Transgenice mice overexpressing this enzyme in skeletal muscle and adipose tissue show an insulin resistance phenotype (4,5). GFAT2, an isoenzyme of GFAT1, was later identified (6, 7). Studies show that the regulation of GFAT2 is different from that of GFAT1, suggesting differential regulation of the hexosamine pathway in different tissues (7).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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