All lanes : Anti-c-Myc (phospho S62) antibody [EPR17924] (ab185656) at 1/2000 dilution

Lane 1 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
Lane 2 : C6 (Rat glial tumor cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 48 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human endometrium cancer tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear staining on cancer cells of Human endometrial cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling c-Myc (phospho S62) with ab185656 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing nuclear staining on HeLa cells.
The staining decreased after blocking with phospho peptide (100μg/ml) overnight.

The control peptide is a non-phospho peptide.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab185656 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear staining on rat testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling c-Myc (phospho S62) with purified ab185656 at 1/150 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

All lanes : Anti-c-Myc (phospho S62) antibody [EPR17924] (ab185656) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) treated with Lambda Phosphatase whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 48 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-c-Myc (phospho S62) antibody [EPR17924] (ab185656) at 1/5000 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 200nM Calyculin A and 1uM Okadaic Acid for 60 minutes.

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 48 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear and cytoplasmic staining on mouse spleen is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

c-Myc (phospho S62) was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 200nM TPA for 10 minutes with ab185656 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab185656 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1500 dilution.

Lane 1: HeLa whole cell lysate treated with 200nM TPA for 10 minutes,10 µg (Input).

Lane 2: ab185656 IP in HeLa whole cell lysate treated with 200nM TPA for 10 minutes.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185656 in HeLa whole cell lysate treated with 200nM TPA for 10 minutes.

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

Dot blot analysis of c -Myc (phospho T58) peptide (Lane 1), c-Myc non-phospho peptide (a control peptide for c-Myc phospho T58) (Lane 2), c-Myc (phospho S62) peptide (Lane 3), and c-Myc non-phospho peptide (a control peptide for c-Myc phospho S62) (Lane 4), labeled using ab185656 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Lanes 1, 2 and 4 are control peptides, lane 3 contains the immunogen peptide.

Exposure time=3 minutes.