All lanes : Anti-p53R2 + RRM2 antibody [EPR20651] (ab209995) at 1/10000 dilution

Lane 1 : His-tagged human RRM2 recombinant protein, 10 ng
Lane 2 : His-tagged human p53R2 recombinant protein, 10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 38,39 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling RRM2/RRM2B with ab209995 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and weak nucleus staining on epithelial cells of human colon (PMID: 23002339). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

All lanes : Anti-p53R2 + RRM2 antibody [EPR20651] (ab209995) at 1/1000 dilution

Lane 1 : Human ovary cancer at 20 µg
Lane 2 : Human thyroid cancer at 20 µg
Lane 3 : Rat brain at 20 µg
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Lane 5 : U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051))

Predicted band size: 45 kDa
Observed band size: 41,45 kDa why is the actual band size different from the predicted?

Blocking: 5% NFDM/TBST.

Exposure times: Lanes 1-3: 3 minutes; Lanes 4-5: 15 seconds.

The MW of RRM2 and p53R2 observed is consistent with the literature (PMID 14661056; PMID 15670581; PMID 25213022).

All lanes : Anti-p53R2 + RRM2 antibody [EPR20651] (ab209995) at 1/1000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 3 : RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051))

Predicted band size: 45 kDa
Observed band size: 41,45 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The MW of RRM2 and p53R2 observed is consistent with the literature (PMID 14661056; PMID 15670581; PMID 25213022).

Anti-p53R2 + RRM2 antibody [EPR20651] (ab209995) at 1/1000 dilution + 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051))

Predicted band size: 45 kDa
Observed band size: 41,45 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The MW of RRM2 and p53R2 observed is consistent with the literature (PMID 14661056; PMID 15670581; PMID 25213022).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling RRM2/RRM2B with ab209995 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse spleen (PMID: 25838540). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

Immunofluorescent analysis of 100% methanol-fixed HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RRM2+ p53R2 with ab209995 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on HeLa cells (PMID: 194169810).

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 100% methanol-fixed NIH/3T3 (mouse embryonic fibroblast) cells labeling RRM2+ p53R2 with ab209995 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on NIH/3T3 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 80% methanol fixed, 0.1% Tween-20 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling p53R2 + RRM2 with ab209995 at 1/500 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.