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Technical Data Sheet

AL9001

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A431 Calyculin A Control
Lysate
Price
Size

$75
100 μl

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Western blot analysis of A431 cells (20 µg/lane) serum starved overnight (lanes 1 & 3) and Calyculin A (100 nM) treated for 30 minutes (lanes 2 & 4). The blot was probed with anti-phospho-Akt (Thr-34) (lanes 1 & 2) or anti-Akt (lanes 3 & 4).

Application
Dilution


WB
20 μl/lane



End user should determine optimal dilution for their particular applications and experiments.Western blot membranes were incubated with diluted antibody in 5% non-fat milk, PBS, 0.04% Tween20 for 1hour at room temperature.
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Background
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.

Buffer and Storage
Cell Lysates are supplied at a concentration of 1 mg/ml in electrophoresis sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, 0.9% β-mercaptoethanol). Store at –20°C. Do not boil or dilute. Stable for 1 year.
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AL9101
A431 + Calyculin A (30min) Lysate

AL9201
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AL9301
A431 + EGF (5 min) Lysate

AL9401
A431 Pervanadate Control Lysate

AL9501
A431 + Pervanadate Lysate

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