Calyculin A, protein phosphatase inhibitor (ab141784)
Key features and details
- Potent, selective and cell-permeable protein phosphatase inhibitor
- CAS Number: 101932-71-2
- Purity: > 99%
- Soluble in ethanol and in DMSO
- Form / State: Solid
- Source: Discodermia calyx
Overview
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Product name
Calyculin A, protein phosphatase inhibitor -
Description
Potent, selective and cell-permeable protein phosphatase inhibitor -
Biological description
Potent, selective and cell-permeable protein phosphatase inhibitor (IC50 values are 2 and 1 nM for PP1 and PP2A, respectively). Highly selective over PP2B and PP2C. Active in vivo and in vitro.
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Purity
> 99% -
CAS Number
101932-71-2 -
Chemical structure
Properties
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Chemical name
[(2R,3R,5R,7R,8S,9S)-2-[(1S,3S,4S,5R,6R,7E,9E,11E,13Z)-14-Cyano-3,5-dihydroxy-1-methoxy-4,6,8,9,13-pentamethyltetradeca-7,9,11,13-tetraenyl]-9-[(E)-3-[2-[(2S)-4-[[(2S,3S,4S)-4-(dimethylamino)-2,3-dihydroxy-5-methoxypentanoyl]amino]butan-2-yl]-1,3-oxazol-4-yl]prop-2-enyl]-7-hydroxy-4,4,8-trimethyl-1,10-dioxaspiro[4.5]decan-3-yl] dihydrogen phosphate -
Molecular weight
1009.18 -
Molecular formula
C50H81N4O15P -
PubChem identifier
5311365 -
Storage instructions
Store at -20°C. Store under desiccating conditions. The product can be stored for up to 12 months. -
Solubility overview
Soluble in ethanol and in DMSO -
Handling
Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20°C. Generally, these will be useable for up to one month. Before use, and prior to opening the vial we recommend that you allow your product to equilibrate to room temperature for at least 1 hour.
Toxic, refer to SDS for further information.
Need more advice on solubility, usage and handling? Please visit our frequently asked questions (FAQ) page for more details.
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SMILES
CC1C(CC2(C(C(C(O2)C(CC(C(C)C(C(C)C=C(C)C(=CC=CC(=CC#N)C)C)O)O)OC)OP(=O)(O)O)(C)C)OC1CC=CC3=COC(=N3)C(C)CCNC(=O)C(C(C(COC)N(C)C)O)O)O -
Source
Discodermia calyx
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Research areas
Images
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Functional Studies - Calyculin A, protein phosphatase inhibitor (ab141784)Calyculin A inhibits the growth of breast cancer epithelial MCF7 cells. Cells were incubated with different concentrations of Calyculin A (ab141784) for four days. Cell number was measured using the methylene blue method. The number of cells was normalized with respect to the control (100%) and plotted against Calyculin A concentrations.
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Immunocytochemistry/ Immunofluorescence - Calyculin A, protein phosphatase inhibitor (ab141784)Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling NF-kB p65 (phospho S276) with ab183559 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).
The expression increased on HeLa cells after treatment with Calyculin A (ab141784 100ng/ml, 10min) then TNF-a (20ng/ml, 5min).
The nuclear counter stain is DAPI (blue).
Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.
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Western blot - Calyculin A, protein phosphatase inhibitor (ab141784)All lanes : Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (ab226821) at 1/1000 dilution
Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) grown in serum-free media overnight, whole cell lysate
Lane 2 : HeLa grown in serum-free media overnight, then treated with 100 ng/ml Calyculin A (ab141784) for 15 minutes, followed by Calyculin A removal and treatment with 100 ng/ml BMP2 for 30 minutes, whole cell lysate
Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) grown in serum-free media overnight, whole cell lysate
Lane 4 : NIH/3T3 cultured in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Developed using the ECL technique.
Observed band size: 60 kDa why is the actual band size different from the predicted?Exposure time:
Lanes 1 and 2: 3 minutes.
Lanes 3 and 4: 30 seconds.Blocking/Dilution buffer: 5% NFDM/TBST.
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Immunoprecipitation - Calyculin A, protein phosphatase inhibitor (ab141784)NF-kB p65 (phospho S276) was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates, treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min, with ab183559 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183559 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min, 10 μg (Input).
Lane 2: ab183559 IP in HeLa whole cell lysate treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab183559 in HeLa whole cell lysate treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.
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Western blot - Calyculin A, protein phosphatase inhibitor (ab141784)All lanes : Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559) at 1/1000 dilution
Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes
Lane 3 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes, then treated with Alkaline Phosphatase for 1 hour
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
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Western blot - Calyculin A, protein phosphatase inhibitor (ab141784)All lanes : Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-29] (ab214423) at 1/1000 dilution
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) grown in serum-free media overnight, whole cell lysate
Lane 2 : HeLa grown in serum-free media overnight, then treated with 100ng/ml Calyculin A (ab141784) for 15 minutes, Calyculin A was removed, followed by treatment with 100ng/ml BMP2 for 30 minutes, whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) grown in serum-free media overnight, whole cell lysate
Lane 4 : NIH/3T3 grown in serum-free media overnight, then treated with 50ng/ml BMP2 for 30 minutes, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Observed band size: 60 kDa why is the actual band size different from the predicted?
Exposure time: 10 secondsBlocking/Dilution: 5% NFDM/TBST.
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Western blot - Calyculin A, protein phosphatase inhibitor (ab141784)All lanes : Anti-AS160 (phospho T642) antibody [EPR2733(2)] (ab131214) at 1.12 µg/ml
Lane 1 : HEK-293 (human embryonic kidney epithelial cell) grown in serum free media overnight whole cell lysate
Lane 2 : HEK-293 grown in serum free media overnight, then treated with 100nM Calyculin A (ab141784) for 50min and then 100ng/ml Insulin was added for the last 20min, whole cell lysate
Lane 3 : HEK-293 grown in serum free media overnight, then treated with 100nM Calyculin A (ab141784) for 50min and then 100ng/ml Insulin was added for the last 20min, whole cell lysate. Then the membrane was incubated with alkaline phosphatase
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilutionBlocking and diluting buffer: 5% NFDM/TBST.
