Immunohistochemical analysis of paraffin-embedded human thyroid gland cancer using anti-ERK1(pT202/pY204)/ERK2(pT185/pY187) (ab32538) at dilution of 1:50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of 4% paraformaldehyde A431+-EGF(100ng/ml,5min) labelling Erk1 (pT202/pY204) + Erk2 (pT185/pY187) with ab32538 at dilution of 1/200. The secondary antibody used was Alexa Fluor^® 488 Goat-Anti-Rabbit IgG (ab150077) at dilution of 1/400. The counter stain was done with DAPI (blue).

Lane 1 : Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [E337] (ab32538) at 1/500 dilution
Lane 2 : Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [E337] (ab32538) at 1/2000 dilution
Lane 3 : Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [E337] (ab32538) at 1/10000 dilution

All lanes : A431 treated with EGF for 10 minutes

Lysates/proteins at 0.1 µg/ml per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 42 , 44 kDa
Observed band size: 42.44 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

First-antibody diluted with 1% BSA.

 

THP1 cells were incubated at 37°C for 3 minutes with vehicle control (0 μM) and different concentrations of ZK 756326 (ab120814). Increased expression of ERK1 (phospho T202 + Y204 ) + ERK2 (phospho T185 + Y187) (ab32538) in THP1 cells correlates with an increase in ZK 756326 concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 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 5% BSA before being incubated with ab32538 at 1/500 dilution and ab17942 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.

Overlay histogram showing HeLa cells stained with ab32538 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32538, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.