Anti-RNF31/HOIP antibody

• WB

Lab

Western blot - Anti-RNF31/HOIP antibody (AB46322)

Western blot : Anti-RNF31 antibody (ab46322) staining at 1/250 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab46322 was shown to bind specifically to RNF31. A band was observed at 130 kDa in wild-type A549 cell lysates with no signal observed at this size in RNF31 knockout cell line. To generate this image, wild-type and RNF31 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-RNF31/HOIP antibody (ab46322) at 1/250 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

RNF31 knockout A549 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 120 kDa

Observed band size: 130 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RNF31/HOIP antibody (AB46322)

ICC/IF image of ab46322 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab46322, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) Hek293 cells at 5µg/ml.

• WB

AbReview63064****

Western blot - Anti-RNF31/HOIP antibody (AB46322)

Western blot analysis of A549 cell lysate (100μg/lane) RNF31/HOIP with ab46322 at 1/1000. A HRP-conjugated Goat anti-rabbit polyclonal (1/10000) was used as the secondary antibody.

lane 1-2 : siCT A549
lane 3-4 : siHOIP A549

All lanes:

Western blot - Anti-RNF31/HOIP antibody (ab46322)

Predicted band size: 120 kDa

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Exposure time: 2min

This image is courtesy of an anonymous Abreview

• WB

Project3281****

Western blot - Anti-RNF31/HOIP antibody (AB46322)

All lanes:

Western blot - Anti-RNF31/HOIP antibody (ab46322) at 1/250 dilution

All lanes:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 120 kDa

Observed band size: 105 kDa,116 kDa,50 kDa,60 kDa

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• WB

Lab

Western blot - Anti-RNF31/HOIP antibody (AB46322)

Western blot : Anti-RNF31 antibody (ab46322) staining at 1/250 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab46322 was shown to bind specifically to RNF31. A band was observed at 130-140 kDa in wild-type A549 cell lysates with no signal observed at this size in RNF31 knockout cell line. To generate this image, wild-type and RNF31 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-RNF31/HOIP antibody (ab46322) at 1/250 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

RNF31 knockout A549 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false