Anti-RBM3 抗体 [EPR6061(2)]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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.

• WB

Lab

Western blot - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

Western blot : Rabbit Monoclonal[EPR6061(2)] to RBM3 ab134946 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at kDa in Wild-type HeLa ab271142 cell lysates with no signal observed at this size in RBM3 knockout HeLa ab274940 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-RBM3 antibody [EPR6061(2)] (ab134946) at 1/1000 dilution

Lane 1:

Wild-type HeLa ab271142 at 20 µg

Lane 2:

RBM3 knockout HeLa ab274940 at 20 µg

Lane 3:

Wild-type HEK-293T at 20 µg

Lane 4:

RBM3 knockout HEK-293T at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 17 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

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:

Western blot - Anti-RBM3 antibody [EPR6061(2)] (ab134946) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

RBM3 Knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human RBM3 knockout HEK-293T cell line (<a href='/products/cell-lines/human-rbm3-knockout-hek-293t-cell-line-ab266663'>ab266663</a>)

Lane 3:

Hela cell lysate at 20 µg

Lane 4:

LnCap cell lysate at 20 µg

Predicted band size: 17 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-RBM3 antibody [EPR6061(2)] (ab134946) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 5:

PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 17 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-RBM3 antibody [EPR6061(2)] (AB134946)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.