Recombinant Anti-MMP14 antibody [EP1264Y] - Low endotoxin, Azide free (ab168726)

This data was developed using the same antibody clone in a different buffer formulation (ab51074).

Lanes 1 - 3: Merged signal (red and green). Green - ab51074 observed at 54 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab51074 was shown to react with MMP14 in A431 wild-type cells in Western blot. Loss of signal was observed when MMP14 knockout sample was used. A431 wild-type and MMP14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% Milk in TBS-T (0.1% Tween®) before incubation with ab51074 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Flow cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell, Left) / HT-1080 (Human fibrosarcoma epithelial cell, Right) cells labeling MMP14 with ab51074 at 1/200 dilution (0.1 μg) (red). Goat anti-rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Rabbit monoclonal IgG (ab172730) / black was used as the isotype control. Cells incubated with secondary antibody only (blue) was used as the unlabeled control. Gated on viable cells.

Positive control (Right panel): HT-1080 cells.

Negative control (Left panel): MCF7 cells.

Ab51074 staining MMP14 in HT-1080 (human fibrosarcoma epithelial cell) cells by ICC/IF (Immunocytochemistry/Immunofluorescence).

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at 1/1000 dilution (0.2 μg/ml). An Alexa Fluor^® 488 Goat anti-rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution (2 μg/ml). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) was used as the counterstain antibody at 1/200 dilution (2.5 μg/ml). DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic and weakly membranous staining in HT-1080 cell line.

Negative control (bottom panels): MCF7 PMID: 19208838.

ab51074 (purified) at 1/20 dilution (2 µg) immunoprecipitating MMP14 in A431 (human epidermoid carcinoma) whole cell lysate.

Lane 1: A431 whole cell lysate 10ug
Lane 2: ab51074 + A431 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab51074 in A431 whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51074).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium carcinoma tissue sections labeling MMP14 with purified ab51074 at 1/100 dilution (1.7 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counterstain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution.

PBS instead of the primary antibody was used as the negative control (inset).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51074).

ab51074 (unpurified) at 1/500 staining human kidney tissue sections by IHC-P.

The tissue was formaldehyde fixed and a heat mediated antigen retrieval step (in Tris/EDTA) was performed. The tissue was then blocked with serum and incubated with the primary antibody. A biotinylated donkey anti-rabbit IgG was used as the secondary.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51074).