Phorbol 12-myristate 13-acetate (PMA), PKC activator

• Chemical Structure

Lab

Chemical Structure - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

2D chemical structure image of ab120297, Phorbol 12-myristate 13-acetate (PMA), PKC activator

• FuncS

Unknown

Functional Studies - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Serum starved HeLa cells were incubated at 37°C for 60 minutes with vehicle control (0 μM) and different concentrations of phorbol 12-myristate 13-acetate (PMA) (ab120297) in DMSO. Increased expression of PKC mu (phospho S916) (ab81218) correlates with an increase in phorbol 12-myristate 13-acetate (PMA) concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab81218 at 1 μg/ml and ab8227 at 1 μg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells, untreated or treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours, then treated with 100ng/ml lipopolysaccharide (LPS) for 7 hours, with 1 μg/ml Brefeldin A (BFA) added after 4 hours, labeling MCP1 with ab214819 at 1/50 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 treated cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt

Lab

Flow Cytometry - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20-permeabilized THP-1 (human monocytic leukemia cell line) cell line, treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h (Right) / Untreated control (Left) labeling MCP1 with ab214891 at 1/500 dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• sELISA

Unknown

Sandwich ELISA - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Sandwich ELISA - IFN gamma Human ELISA Kit (ab46025)

Jurkat were stimulated for 48 hours with 50 ng x mL-1 of PMA (ab120297) and 1 μM Ionomycin (ab120116) and PBMCs were stimulated for 48 hours with 2 % PHA-M (LifeTechnologies). Cell free supernatants were tested, showing results after background signal was subtracted (duplicates +/- SD).

• WB

Supplier Data

Western blot - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23129404 and PMID : 6423641).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Interferon gamma antibody [RM1245] (<a href='/products/primary-antibodies/interferon-gamma-antibody-rm1245-ab322926'>ab322926</a>) at 1/1000 dilution

Lane 1:

Untreated No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate at 20 µg

Lane 2:

No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (ab120297) and 3uM Ionomycin (<a href='/products/biochemicals/ionomycin-ca2-salt-ca2-ionophore-ab120116'>ab120116</a>) for 5 hours, 300 ng/ml BFA was added for last 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 17 kDa,19 kDa,23 kDa,36 kDa

false

Exposure time: 92s

• IP

Lab

Immunoprecipitation - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

CD69 was immunoprecipitated from 0.35 mg Daudi (human Burkitt's lymphoma lymphoblast) treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate with ab233396 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab233396 at 1/5,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.

Lane 1 : Daudi (human Burkitt's lymphoma lymphoblast) treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate 10 μg (Input).
Lane 2 : ab233396 IP in Daudi treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab233396 in Daudi treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate (-).

Blocking/Dilution buffer : 5% NFDM/TBST.

false

Exposure time: 30s

• sELISA

Unknown

Sandwich ELISA - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Sandwich ELISA - IL-1ra (Interleukin-1ra) Mouse ELISA Kit (ab113348)

IL-1Ra detected in supernatants from RAW 246.7 control cells (C) or cells stimulated for 24 hours with 50 ng x mL-1 of PMA (ab120297) (P), or 24 hours with PMA and 1 ug x mL-1 of LPS (Sigma) (P+L) for the last 6 hours. Results shown after background signal was subtracted (duplicates +/- SD).

• sELISA

Unknown

Sandwich ELISA - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Sandwich ELISA - IL-6 (Interleukin-6) Mouse ELISA Kit (ab100712).

IL-6 detected in supernatants from RAW 246.7 control cells (C) or cells stimulated for 24 hours with 50 ng x mL-1 of PMA (ab120297) (P), or 24 hours with PMA and 1 ug x mL-1 of LPS (Sigma) (P+L) for the last 6 hours. Results shown after background signal was subtracted (duplicates +/- SD).

• sELISA

Unknown

Sandwich ELISA - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

Sandwich ELISA - TNF alpha Human ELISA Kit (ab100654)

TNFa detected in supernatants from control cells (C) or cells stimulated for 24 hours with 50 ng x mL-1 of PMA (ab120297) (P), and PMA with the addition of 1 ug x mL-1 of LPS (Sigma) (P+L) for the last 6 hours. Results shown after background signal was subtracted (duplicates +/- SD).

• IP

Lab

Immunoprecipitation - Phorbol 12-myristate 13-acetate (PMA), PKC activator (AB120297)

MCP1 was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate with ab214819 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab214819 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate 10 μg (Input).

Lane 2 : ab214819 IP in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab214819 in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA, ab120297) for 24h, then treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1μg/ml Brefeldin A (BFA) for another 3h whole cell lysate.

Blocking and dilution buffer : 5% NFDM/TBST.

false

Exposure time: 30s