All lanes : Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 250 ng/ml Anisomycin for 30 minutes whole cell lysate
Lane 2 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) 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: 36 kDa
Observed band size: 40,45,48 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/dilution buffer: 5% NFDM/TBST.

Per the blast, ab178858 could recognize JunD (phospho Ser100) with 100% homology. Multi-bands are due to c-Jun (phospho Ser73) & JunD (phospho Ser100).

Dot blot analysis of JunD (phospho S100) + c-Jun (phospho S73) peptide (Lane 1) and non-phospho peptide (Lane 2) labeled using ab178858 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.

Exposure time: 3 minutes.

 

Lanes 1 & 4-5 : Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)
Lanes 2-3 : Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858) at 1/10000 dilution

Lanes 1 & 3 & 5 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lanes 2 & 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 250 ng/ml Anisomycin for 30 minutes 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: 36 kDa
Observed band size: 40,45 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 250 ng/ml Anisomycin for 30 minutes whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 250 ng/ml Anisomycin for 30 minutes, whole cell lysate treated with Alkaline Phosphatase

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: 36 kDa
Observed band size: 40,45 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858) at 1/10000 dilution

Lane 1 : NIH/3T3 (Mouse embryo fibroblast cell line) treated with 250 ng/ml Anisomycin for 30 minutes whole cell lysate
Lane 2 : Untreated NIH/3T3 (Mouse embryo fibroblast cell line) 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: 36 kDa
Observed band size: 40,45 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/dilution buffer: 5% NFDM/TBST.

Per the blast, ab178858 could recognize JunD (phospho Ser100) with 100% homology. Multi-bands are due to c-Jun (phospho Ser73) & JunD (phospho Ser100).

All lanes : Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858) at 1/1000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) 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: 36 kDa
Observed band size: 40,45 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/dilution buffer: 5% NFDM/TBST.

Per the blast, ab178858 could recognize JunD (phospho Ser100) with 100% homology. Multi-bands are due to c-Jun (phospho Ser73) & JunD (phospho Ser100).

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling JunD (phospho S100) + c-Jun (phospho S73) with ab178858 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on lymphocytes and endothelial cells of Human tonsil is observed. Counter stained with Hematoxylin.

Negative Control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling JunD (phospho S100) + c-Jun (phospho S73) with ab178858 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on spermatogoniums and Leydig cells of mouse testis is observed. Counter stained with Hematoxylin.

Negative Control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling JunD (phospho S100) + c-Jun (phospho S73) with ab178858 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on bile duct epithelial cells while no staining on hepatocytes of rat liver is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

JunD (phospho S100) + c-Jun (phospho S73) were immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma), treated with 250ng/ml Anisomycin for 30 minutes, whole cell extract with ab178858 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab178858 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.
Lane 1: HeLa, treated with 250ng/ml Anisomycin for 30 minutes, whole cell extract 
Lane 2: PBS.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.

 

Per the blast, ab178858 could recognize JunD (phospho Ser100) with 100% homology. Multi-bands are due to c-Jun (phospho Ser73) & JunD (phospho Ser100)