Western blot analysis of extract from human kidney using Glutaminase-1/GLS1 Antibody.
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 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
Glutaminase-1/GLS1 Antibody recognizes endogenous levels of total glutaminase-1/GLS1 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly116 of human glutaminase-1/GLS1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Glutaminase catalyzes the conversion of glutamine to glutamate, the first and rate-limiting step of glutaminolysis (1). Both kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2) are found in mammals (2). GLS1-mediated glutathione synthesis plays an essential role in redox homeostasis and contributes to increased survival of postimplantation bone cells preconditioned to the hypoxic and ischemic environment in the bone defect site (3). In addition, KEAP1–NRF2-mutant LUAD (KRAS-mutant lung adenocarcinoma) tumors are dependent on increased glutaminolysis (1). Furthermore, recent studies showed higher glutaminolysis and glucose production from glutamine in human primary hepatocytes with GLS2 gain-of-function missense mutations (4). These findings suggest GLS1 and GLS2 as potential targets in the therapy of bone regeneration and in the treatments to diseases such as cancer and hyperglycemia, respectively (1, 3-4).
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