Anti-MMP14 antibody [EP1264Y] - BSA and Azide free

• Flow Cyt

Lab

Flow Cytometry - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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

Flow cytometry overlay histogram showing wild-type A431 (green line) and A431 MMP14 knockout stained with ab51074 (magenta line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab51074) (1x 10⁶in 100μl at 1.0 μg/ml (1/2070)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type A431 - black line, A431 MMP14 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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

• WB

Lab

Western blot - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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

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

All lanes:

Western blot - Anti-MMP14 antibody [EP1264Y] (<a href='/en-us/products/primary-antibodies/mmp14-antibody-ep1264y-ab51074'>ab51074</a>) at 1/2000 dilution

All lanes:

Human lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 66 kDa

false

Exposure time: 40s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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 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.

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

• IP

Unknown

Immunoprecipitation - Anti-MMP14 antibody [EP1264Y] - BSA and Azide free (AB271840)

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.

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

All lanes:

Immunoprecipitation - Anti-MMP14 antibody [EP1264Y] (<a href='/en-us/products/primary-antibodies/mmp14-antibody-ep1264y-ab51074'>ab51074</a>)

Predicted band size: 65 kDa

Observed band size: 66 kDa

false