10X Blocking Buffer

• WB

Unknown

Western blot - 10X Blocking Buffer (AB126587)

Primary :

All Lanes : top membranes with Anti AMPK pThr172 + Thr183 (ab133448) at 1/1000. Bottom membranes with Anti AMPKa (ab110036) at 1/1000

Lane 1 = AMPKa1 protein

Lane 2 = C2C12 cells treated with AICAR

Lane 3 = untreated C2C12 cells

Secondary : Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) and Goat polyclonal to Mouse IgG – H&L – Pre-Adsorbed (HRP) at 1 : 10,000 developed using the ECL technique.

Performed under reducing conditions (50mM DTT – Sample heated at 60°C). Predicted band size for AMPKa : 64 kDa Observed band size for AMPKa : 64 kDa

Left membrane All blocking steps = 1X blocking reagent ab126587 diluted in PBST

Right membrane All blocking steps = 5% Milk in PBST Exposure time : 5 minutes

false

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - 10X Blocking Buffer (AB126587)

HeLa cells were untreated or treated with 1mM Deferoxamine (DFO) for 24h and fixed with paraformaldehyde for imaging by fluorescent microscopy. Cells were blocked and stained with 1X blocking buffer (ab126587). Unpurified ab51608 was used at 1 : 500. DAPI was used to label the nucleus. HIF1 alpha staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus.