- MA1-141 - Invitrogen Antibodies COL1A2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of collagen I was performed by loading the indicated amounts of bovine collagen I onto a 4-12% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA in TBST for at least 1 hour. The membrane was probed with a collagen I monoclonal antibody (Product # MA1-141) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Direct ELISA analysis of collagen I was performed by coating 100 µL per well of bovine collagen I in twelve replicates at 50, 25, 12.5, 6.25, 3.12, 1.56, 0.781 and 0 µg/mL across a clear, 96-well plate and incubating overnight at room temperature. The coating solution was decanted and the plate was blocked with 5% BSA in TBST for at least 2 hours. The plate was washed with TBST and then incubated with 100 µL per well of collagen I monoclonal antibody (Product # MA1-141) at 50 µg/mL (red line), 10 µg/mL (blue line), 2 µg/mL (green line) and 0.4 µg/mL (orange line) in blocking buffer for 1 hour at room temperature. The plate was washed and incubated with 100 µL per well of HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) in all test wells at 1:10,000 for 30 minutes at room temperature. The plate was again washed in TBST and detection was performed using ultra TMB substrate (Product # 34028) for 30 minutes at room temperature in the dark. The reaction was then stopped with 0.16M sulfuric acid and absorbance was read at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Collagen I was performed on porcine skin tissue. To expose target proteins, antigen retrieval was performed by microwaving tissues for 8-15 minutes in 10mM sodium citrate buffer (pH 6.0). Following antigen retrieval, tissues were blocked in 3% hydrogen peroxide-methanol for 15 min at room temperature, washed with deionized water and PBS, and then probed with a Collagen I monoclonal antibody (Product # MA1-141) diluted 1:20 in 3% BSA-PBS (right panel) or incubated with buffer alone not containing primary antibody as a negative control (left panel), overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Collagen I was performed on frozen human breast carcinoma tissue. Tissues were probed with a Collagen I monoclonal antibody (Product # MA1-141, right panel) or mouse IgG isotype control (left panel) at a dilution of 1:500 for 1 hour at room temperature. Tissues were washed, and detection was performed using an HRP-conjugated universal detection reagent followed by DAB substrate. Tissues were counterstained and prepped for mounting before visualization by light microscopy.

Supportive validation

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Direct ELISA analysis of collagen I was performed by coating 100 µL per well of bovine collagen I in twelve replicates at 50, 25, 12.5, 6.25, 3.12, 1.56, 0.781 and 0 µg/mL across a clear, 96-well plate and incubating overnight at room temperature. The coating solution was decanted and the plate was blocked with 5% BSA in TBST for at least 2 hours. The plate was washed with TBST and then incubated with 100 µL per well of collagen I monoclonal antibody (Product # MA1-141) at 50 µg/mL (red line), 10 µg/mL (blue line), 2 µg/mL (green line) and 0.4 µg/mL (orange line) in blocking buffer for 1 hour at room temperature. The plate was washed and incubated with 100 µL per well of HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) in all test wells at 1:10,000 for 30 minutes at room temperature. The plate was again washed in TBST and detection was performed using ultra TMB substrate (Product # 34028) for 30 minutes at room temperature in the dark. The reaction was then stopped with 0.16M sulfuric acid and absorbance was read at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 Notum inhibited liver fibrosis gene expression in LX-2 cells with HBV exposure. a Cell co-culture model. b LX-2 cells were co-cultured with Hep AD38 cells and transferred with Notum. alpha-SMA, TIMP-1 and Col 1A1 mRNA expressions in LX-2 cells were measured by qPCR. c , d alpha-SMA, TIMP-1 and Col 1A1 protein expressions in LX-2 cells were measured by Western-blot. * P < 0.05 compared with mock. All experiments were tried 3 times

MA1-141