Western blot analysis of extracts from U-2 OS, MOLT-4 and Jurkat cells using BCAT1 Antibody.
Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing either Myc/DDK-tagged full-length human BCAT1 (hBCAT1-Myc/DDK; +) or Myc/DDK-tagged full-length human BCAT2 (hBCAT2-Myc/DDK; +), using BCAT1 Antibody (upper), DYKDDDDK Tag (9A3) Mouse mAb #8146 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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 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
BCAT1 Antibody recognizes endogenous levels of total BCAT1 protein. This antibody cross-reacts with BCAT2.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human BCAT1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
BCAT1 and BCAT2 are cytosolic and mitochondrial branched chain aminotransferases, respectively (1,2). Research studies have implicated BCAT1 in distant metastasis in patients with advanced colorectal cancer (3). Disruption of BCAT2 in mice leads to higher levels of plasma branched-chain amino acids, reduced adiposity and body weight, and increased energy expenditure, suggesting its role in regulating insulin sensitivity (4).
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