Recombinant Anti-RBM3 antibody [EPR6061(2)] (ab134946)

All lanes : Anti-RBM3 antibody [EPR6061(2)] (ab134946) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : RBM3 Knockout HEK-293T cell lysate
Lane 3 : Hela cell lysate
Lane 4 : LnCap cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 17 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab134946 observed at 18 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab134946 was shown to react with RBM3 in wild-type HEK-293T cells in Western blot with loss of signal observed in RBM3 knockout cell line ab266663 (RBM3 knockout cell lysate ab258170). Wild-type HEK-293T and RBM3 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab134946 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-RBM3 antibody [EPR6061(2)] (ab134946) at 1/1000 dilution (Purified)

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysate

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

Predicted band size: 17 kDa

We are unsure how to define the extra bands.

Immunocytochemistry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling RBM3 with Purified ab134946 at 1/100 dilution (1.32 µg/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 dilution (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/mL). DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue sections labeling RBM3 with Purified ab134946 at 1/300 dilution (0.44 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling RBM3 with Purified ab134946 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling RBM3 with Purified ab134946 at 1/300 dilution (0.44 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human astrocytoma tissue sections labeling RBM3 with Purified ab134946 at 1/300 dilution (0.44 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Equilibrium disassociation constant (K_D)
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