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The galactose treated HeLa lysate was generated after switching HeLa cells from glucose to galactose in the presence of dialyzed serum with continued growth for 12 passages prior to harvesting the cells (DMEM containing 10% dialized FCS, 6mM L-glutamine, 1mM sodium pyruvate, 1 g/L Sodium bicarbonate, 10mM HEPES, and 5mM Galactose). The glucose HeLa lysate was generated from cells grown in HGDMEM containing 10% qualified FCS, 1mM Sodium Pyruvate, 1.5 g/L Sodium bicarbonate, and 4mM L-Glutamine.
The combination of the galactose treated and untreated set is therefore designed for use as a western blot control when studying protein extracts for mitochondria metabolism and metabolic changes based on fuel adaptations.
Concentration: HeLa Galactose lysate, 200 µg at 2.0 mg/mL
HeLa Glucose lysate, 200 µg at 2.0 mg/mL
Galactose is a non fermentable carbon source frequently used to measure the effect of fuel on multiple protein metabolic markers. Growth of cells in-vitro with galactose instead of glucose, results in activation of the Galactose-Glucose interconversion pathway. This is a high energy phosphate consuming pathway. Due to the high energy cost and the inefficiency by which galactose is converted in-vitro into glucose, the consumption of energy to generate one molecule of glucose from galactose is almost as costly as the amount of energy generated by that molecule of glucose via glycolysis. Carbons from galactose are therefore ultimately converted into glucose which is decarboxylated to a pentose sugar to serve as substrate of the pentose-phosphate shunt.
Under these circumstances, cells are obliged to use glutamine (present in the media) as the main source of carbons for the anaplerosis of the citric acid cycle and therefore the main source of energy. The switch from glycolysis to glutaminolysis in the presence of galactose requires acute and long term proteomic adaptive responses.
|HeLa Galactose Lysate||1 x 200µg|
|HeLa Glucose Lysate||1 x 200µg|
Our Abpromise guarantee covers the use of ab176107 in the following tested applications.
|WB||Use at an assay dependent concentration.
2.0 mg/mL. Load 30 µg per lane. Heat sample at 70°C before loading.
|SDS-PAGE||Use at an assay dependent concentration.|
Lane 1: Marker
Lane 2 : HeLa Glucose Whole Cell Lysate – 30 µg
Lane 3 : HeLa Galactose Whole Cell Lysate – 30 µg
Primary antibodies: Anti- mTOR pSer2448 (ab109268) at 1:1000 dilution.
Anti-Actin (ab1801) at 1:5000 dilution
Secondary: Goat polyclonal to Rabbit IgG (ab97080) – H&L – Pre-Absorbed (HRP) at 1/10000 dilution
Developed using the ECL technique under reducing conditions; Exposure time: 5 mins; Blocking and antibody incubation steps done in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20
Observed mTOR band size: 288kDa
Lane 1: Marker
Lane 2 : HeLa Glucose Whole Cell Lysate – 30µg
Lane 3 : HeLa Galactose Whole Cell Lysate - 30µg
MitoProfile® Pyruvate dehydrogenase (PDH) WB Antibody Cocktail (ab110416) at 6 µg/mL
Anti-actin (ab1801) at 1:5000 dilution
Developed using the ECL technique under reducing conditions; Samples run on a 4-20% gradient gel. Exposure time: 5 mins; Blocking and antibody incubation steps done in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20
Observed E2 band size = 69kDa
Observed E3bp band size = 54kDa
Observed E1α band size = 43kDa
Observed E1β band size = 39kDa
Observed OSCP subunit of ATPsynthase = 22kDa
HeLa cells lysates were loaded at 30 µg per lane on a 4-20% gradient gel, then stained with Optiblot Blue (ab119211) for 2 hours at room temperature.
Gel was destained using nanopure water for 24 hours.
ab176107 has not yet been referenced specifically in any publications.