重组Anti-MMP14抗体[EP1264Y] (ab51074)

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

Lane 1 : A431 wild-type cell lysate
Lane 2 : A431 MMP14 knockout cell lysate
Lane 3 : Caco-2 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.

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.

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 (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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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.