Calyculin A is a serine/threonine phosphatase inhibitor that inhibits the activity of protein phosphatases PP1 and PP2A. Human carcinoma A431 cells treated with calyculin A for 30 minutes can undergo significant threonine phosphorylation, as shown by western blotting using anti-Phospho-Akt (Thr-34), cat.# AP1001, as compared to untreated, control cell lysates.
Confluent cultures of A431 cells were serum starved overnight. Cells were then either left untreated (Cat.# AL9001) or treated with Calyculin A at a final concentration of 100 nM for 30 minutes at 37°C (Cat.# AL9101). Cells were lysed in 1% SDS, 1.0 mM sodium ortho-vanadate, 1 mM sodium fluoride, 10 mM Tris (pH 7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer.
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*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blot mobilities of known proteins with similar MW.
This kit contains: