Recombinant Anti-MMP14 antibody [EP1264Y] (ab51074)

All lanes : Anti-MMP14 antibody [EP1264Y] (ab51074) at 1/2000 dilution

Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : MMP14 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 66 kDa
Observed band size: 54 kDa why is the actual band size different from the predicted?

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.

ab51074 staining MMP14 in wild-type A431 cells (top panel) and MMP14 knockout A431 cells (bottom panel) (ab261890). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab51074 at 0.2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

All lanes : Anti-MMP14 antibody [EP1264Y] (ab51074) at 1/2000 dilution (unpurified)

Lane 1 : Human fetal spleen tissue lysate
Lane 2 : Human lung cancer tissue lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 66 kDa
Observed band size: 60,63 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Diluting buffer and concentration 5% NFDM/TBST

63 kDa: pro-form; 60 kDa: active form.

MCF7 is a MMP14 negative or weakly expressed cell line (PMID: 25977338 and PMID: 19208838).

All lanes : Anti-MMP14 antibody [EP1264Y] (ab51074) at 1/5000 dilution (purified)

Lane 1 : Human fetal spleen lysate
Lane 2 : Human lung cancer lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat spleen lysate
Lane 5 : Human esophagus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 66 kDa
Observed band size: 66 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human endometrial cancer labeling MMP14 with ab51074 at 1/500 (3.26 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Postive staining on the human endometrial tumour stroma cells and weak staining on the tumour cells. The section was incubated with ab51074 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Human endometrial cancer labeling MMP14 with ab51074 at 1/500 (3.26 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human endometrial cancer. The section was incubated with ab51074 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. 

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes.

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).

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, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

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

Intracellular 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 Fluorr^® 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 (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.

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