Anti-RNF5/NG2 antibody [EPR27133-30]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling RNF5/NG2 with ab308066 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab308066.

• IP

Supplier Data

Immunoprecipitation - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

RNF5/NG2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab308066 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308066 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa whole cell lysate Lane 2 : ab308066 IP in HeLa whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab308066 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/30 dilution

All lanes:

HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 19 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

Western blot : Rabbit Monoclonal[EPR27133-30] to RNF5/NG2 ab308066 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 18 kDa in Wild-type A549 cell lysates with no signal observed at this size in RNF5 knockout A549 cell line (ab326022). 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-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human RNF5 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rnf5-knockout-a549-cell-line-ab326022'>ab326022</a>) at 20 µg

Lane 3:

SH-SY5Y at 20 µg

Lane 4:

A-498 at 20 µg

Secondary

All lanes:

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

Predicted band size: 20 kDa

Observed band size: 18 kDa

false

• WB

Supplier Data

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with human RNF185. In Western blot, anti-GST antibody (ab111947) staining at 1/5000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

Lane 1:

Human RNF5 recombinant protein

Lane 2:

Human RNF185 recombinant protein

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human colon tissue lysate at 20 µg

Lane 3:

Human colon cancer tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 19 kDa

true

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (AB308066)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (ab308066) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

293T transfected with siRNA specifically targeting RNF5/NG2 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 19 kDa

true

Exposure time: 180s