Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using ab181140, the same antibody clone in a different buffer formulation.

Intracellular Flow Cytometry analysis of HepG2 (human hepatocellular carcinoma) cells labeling RING2 / RING1B / RNF2 with purified ab181140 at 1/70 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using ab181140, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling RING2 / RING1B / RNF2 with ab181140 at 1/500 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using ab181140, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of HepG2 cells (paraformaldehyde-fixed, 4%) labeling RING2 / RING1B / RNF2 with ab181140 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor® 555) secondary at 1/200 dilution and counter-stained with DAPI (blue).

• WB

Lab

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using ab181140, the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : RING2 / RING1B / RNF2 knockout HAP1 cell lysate (20 μg)

Lane 3 : HeLa cell lysate (20 μg)

Lane 4 : Jurkat cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab181140 observed at 42 kDa. Red - loading control, ab7291, observed at 52 kDa.

ab181140 was shown to recognize RING2 / RING1B / RNF2 when RING2 / RING1B / RNF2 knockout samples were used, along with additional cross-reactive bands. Wild-type and RING2 / RING1B / RNF2 knockout samples were subjected to SDS-PAGE. ab181140 at a dilution of 1/1000 and ab7291 (loading control to alpha Tubulin) at a dilution of 1/10,000 were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] (<a href='/en-us/products/primary-antibodies/ring2-ring1b-rnf2-antibody-epr12245-ab181140'>ab181140</a>)

Predicted band size: 37 kDa

false

• WB

Lab

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using the same antibody clone in a different buffer formulation (ab181140).

Lanes 1- 2 : Merged signal (red and green). Green - ab181140 observed at 42 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab181140 was shown to react with RING2 / RING1B / RNF2 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264845 (CRISPR/Cas9 edited cell lysate ab257640) lane below 42kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and RNF2 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181140 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] (<a href='/en-us/products/primary-antibodies/ring2-ring1b-rnf2-antibody-epr12245-ab181140'>ab181140</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RNF2 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RNF2 (RING2 / RING1B) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rnf2-ring2-ring1b-knockout-hela-cell-line-ab264845'>ab264845</a>)

Predicted band size: 37 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] - BSA and Azide free (AB250376)

This data was developed using ab181140, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-RING2 / RING1B / RNF2 antibody [EPR12245] (<a href='/en-us/products/primary-antibodies/ring2-ring1b-rnf2-antibody-epr12245-ab181140'>ab181140</a>) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 20 µg

Lane 2:

293 cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 37 kDa

false