Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) treated with EGF (100ng/ml, 5min) and treated with Lambda Protein Phosphatase 31℃ for 2h cells labeling Estrogen receptor alpha (phospho S118) with purified ab32396 at 1:200 dilution (8.9μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-Estrogen Receptor alpha (phospho S118) antibody [E91] (ab32396) at 1/1000 dilution (purified)

Lane 1 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : MCF-7 (Human breast adenocarcinoma epithelial cell) treated with epidermal growth factor. Whole cell lysates
Lane 3 : MCF-7 (Human breast adenocarcinoma epithelial cell) treated with epidermal growth factor. Whole cell lysates. Then the membrane was incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 66 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

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

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling Estrogen Receptor alpha (phospho S118) with unpurified ab32396 at 5 μg/ml (1/200 dilution). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077, an AlexaFluor^®488 Goat anti-Rabbit  was used as the secondary antibody at 2 μg/ml (1/1000 dilution). ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain at 2.5 μg/ml (1/200 dilution). DAPI nuclear counterstain.
Confocal image showing the signal increased after EGF (100ng/ml, 5 min) treatment and decreased after Lambda Protein Phosphatase treatment (31°C for 2 hours).

Immunohistochemical staining of (A) untreated and (B) Phosphatase-treated paraffin-embedded human breast adenocarcinoma using unpurified ab32396 at 1:50 dilution.

Dot blot analysis of Lane 1: Estrogen Receptor alpha (pS118) phospho peptide and Lane 2: Estrogen Receptor alpha non-phospho peptide labeling Estrogen Receptor alpha (phospho S118) with unpurified ab32396 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051, Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.

Immunofluorescent staining of (A) untreated and (B) Phosphatase-treated MCF-7 cells using unpurified ab32396.

All lanes : Anti-Estrogen Receptor alpha (phospho S118) antibody [E91] (ab32396) at 1/1000 dilution (unpurified)

Lane 1 : MCF7 cell lysate (untreated).
Lane 2 : MCF7 cell lysate (treated with b-Estradiol and EGF).

Predicted band size: 66 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?