Immunofluorescence staining showing MMP9 (green) and DAPI (blue) in 12 weeks old N-IF and 24αβNOD control mouse livers. Scale bars are 100 μm.

Immunohistochemical staining was performed on liver biopsies fixed in 4% paraformaldehyde and embedded in OCT. The frozen tissues were cut in 5 μm thick sections and stained using primary antibody against matrix metallopeptidase 9 (MMP9) (1:300, Abcam, ab38898). The nuclei were visualized with DAPI. The sections where analyzed using confocal microscopy.

All lanes : Anti-MMP9 antibody (ab38898) at 5 µg/ml

Lane 1 : Recombinant Human MMP9, His tagged (ab82955) at 0.1 µg
Lane 2 : U937 whole cell lysate at 100 µg
Lane 3 : U937 whole cell lysate - treated with PMA and Brefeldin (24 hour treatment) at 100 µg
Lane 4 : Raw 264.7 (Mouse) whole cell lysate at 100 µg
Lane 5 : Raw 264.7 (Mouse) whole cell lysate - treated with LPS (6 hour treatment, 1ug/mL) at 100 µg

Performed under reducing conditions.

ab38898 detects recombinant Human MMP9 running at ~85 kDa, and endogenous full-length MMP9 in LPS-stimulated cells at ~100 kDa. This antibody also detects a band at 90 kDa in U937 PMA-treated cells. 

 

ab38898 was incubated at 5 ug/mL and ab8245 (loading control to GAPDH) was diluted at 0.1 ug/mL and both were incubated for 48 hours at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

ab38898 at a 1/1000 dilution staining mouse heart tissue by Immunohistochemistry (Frozen sections). The tissue was removed from a mouse, rinsed in PBS and slowly frozen in supercooled isopentane. 14um sections were made using a cryostat. The sections were acetone fixed and blocked in 2% BSA prior to incubation with the MMP9 antibody. Goat Anti-Rabbit IgG H&L (Cy3 ®) preadsorbed (ab6939)  was used as the secondary antibody. In the image: red staining = MMP9, blue staining = nuclei, green = gelatinase activity.

All lanes : Anti-MMP9 antibody (ab38898) at 2 µg/ml

Lane 1 : Native human MMP9 protein (Proenzyme, monomer) (ab157344)
Lane 2 : Recombinant Mouse MMP9 protein (ab39309)

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Infrared labelled goat anti-rabbit (green) at 1/20000 dilution

Performed under reducing conditions.

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with anti-MMP9 antibody (ab38898; 2 microgram per mL)  overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-rabbit (green) antibody (diluted 1:20000) for 1 hour at room temperature before imaging.

Anti-MMP9 antibody (ab38898) + Human MMP9

Observed band size: 88,92 kDa why is the actual band size different from the predicted?



Ab38898 detects a band at 92 Kd (pro-form) and a band at 88 Kd (active form). Mouse MMP9 is slightly larger than human MMP9, and the antibody detects a band at about 105 Kd. It is recommend to concentrate samples by Gelatin-agarose affinity chromatography prior to Western blot usage. A recommended starting concentration for Western blots is 1:1000 when using colorimetric substrates such as BCIP/NBT, and 1:5000 for chemiluminescent substrates. Higher concentration of antibody may be needed for non-human samples.