重组Anti-MMP3抗体[EP1186Y] (ab52915)

Western blot: Anti-MMP3 antibody [EP1186Y] (ab52915) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52915 was shown to bind specifically to MMP3. A band was observed at 55-60 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in MMP3 knockout cell line. To generate this image, wild-type and MMP3 knockout U-87 MG 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.

Blocking and diluting buffer: 5% NFDM/TBST.

Immunofluorescence staining of HT-29 cells with purified ab52915 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 100% methanol and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab52915 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Blocking and diluting buffer: 5% NFDM/TBST.

Exposure time: Lane 1 & 2: 10 seconds
                         Lane 3: 3 minutes

Blocking and diluting buffer: 5% NFDM/TBST.

Exposure time:  Lane 1: 3 minutes
                           Lane 2 & 3: 4 seconds

Blocking and diluting buffer: 5% NFDM/TBST

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST