- MA1-188 - Invitrogen Antibodies PGF antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of recombinant human PLGF-1 performed by loading 2 µgs of of protein, and 15 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock (PBS) Blocking Buffer (Product # 37538) for at least 1 hour at room temperature. Recombinant human PLGF-1 was detected at ~14 kD using a PLGF-1 monoclonal antibody (Product # MA1-188) at a concentration of 4 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody (Product # 31439) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Sandwich ELISA analysis of human recombinant PLGF-1 was performed by coating wells of a plate with mouse anti PLGF-1 monoclonal antibody (Product # MA1-188) at a concentration of 2 µg/mL overnight at 4C. The plate was washed 3 times with ELISA Wash Buffer (Product # N503) and blocked with blocking buffer (Product # 37538) for one hour at room temperature. Human recombinant PLGF-1 protein was initially diluted 100 ng/mL and further serially diluted 1:2 for 11 dilutions. 100 µL of diluted human recombinant PLGF-1 protein were added to wells in duplicate and incubated for 2 hours at room temperature. The plate was washed, and then incubated with 100 µL per well of Biotinylated mouse anti PLGF antibody at a concentration of 1 µg/mL for 1 hour at room temperature. The plate was washed and incubated with conjugated Streptavidin-HRP (Product # 21130) at a dilution of 1:10,000 for 1 hour at room temperature, and washed again with ELISA Wash Buffer. The plate was developed by incubating 100 µL per well of 1-Step Ultra TMB substrate (Product # 34028) per well for 20 minutes at room temperature in the dark. The reaction was stopped with 100 µL per well of Stop solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of PLGF-1 proteins were performed on HUVEC cells conditioned media. Antigen-antibody complexes were formed by incubating 1 mL of concentrated HUVEC cells conditioned media with 10 µg of a PLGF-1 monoclonal antibody (Product # MA1-188) overnight on a rocking platform at 4C. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with StartingBlock (PBS) Blocking Buffer (Product # 37538) for at least 1 hour at room temperature. Secreted PLGF-1 was detected at ~24 kD, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody (Product # 31439) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of PLGF-1 proteins were performed on HUVEC cells conditioned media. Antigen-antibody complexes were formed by incubating 1 mL of concentrated HUVEC cells conditioned media with 10 µg of a PLGF-1 monoclonal antibody (Product # MA1-188) overnight on a rocking platform at 4C. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with StartingBlock (PBS) Blocking Buffer (Product # 37538) for at least 1 hour at room temperature. Secreted PLGF-1 was detected at ~24 kD, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody (Product # 31439) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

MA1-188