All lanes : Anti-PCBP2/hnRNP E2 antibody [EPR14859(2)] (ab200835) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 34-39 kDa
Observed band size: 35,39 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Based on sequence analysis, ab200835 recognizes isoforms 1-6 which have corresponding Mw between 34-39kDa.

Blocking/Dilution Buffer: 5% NFDM/TBST.

All lanes : Anti-PCBP2/hnRNP E2 antibody [EPR14859(2)] (ab200835) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat heart lysate
Lane 4 : Rat spleen 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: 34-39 kDa
Observed band size: 35,39 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution Buffer: 5% NFDM/TBST.

All lanes : Anti-PCBP2/hnRNP E2 antibody [EPR14859(2)] (ab200835) at 1/1000 dilution

Lane 1 : Rat heart lysate
Lane 2 : C6 (Rat glial tumor cells) whole cell lysate
Lane 3 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 5 : NIH/3T3 (Mouse embyro fibroblast 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: 34-39 kDa
Observed band size: 35,39 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution Buffer: 5% NFDM/TBST.

All lanes : Anti-PCBP2/hnRNP E2 antibody [EPR14859(2)] (ab200835) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with PCBP2/hnRNP E2 peptide
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with PCBP1 peptide

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: 34-39 kDa
Observed band size: 35,39 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution Buffer: 5% NFDM/TBST.

Based on sequence analysis, ab200835 (PCBP2/hnRNP E2) shares 80% homology with family member PCBP1. The levels of XR were tested in the accompanying blocking experiment.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling PCBP2/hnRNP E2 with ab200835 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear and cytoplasmic staining on HeLa cell line is observed. 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: ab200835 at 1/500 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.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling PCBP2/hnRNP E2 with ab200835 at 1/250 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

PCBP2/hnRNP E2 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate with ab200835 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab200835 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: Jurkat whole cell lysate 10 µg (Input). Lane 2: ab200835 IP in Jurkat whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200835 in Jurkat whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds