- MA1-19178 - Invitrogen Antibodies AFP antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of alpha Fetoprotein was performed on HepG2 whole cell lysates. Cell lysates were mixed with reducing (Lane 1) or non-reducing (Lane 2) Laemmli SDS-PAGE sample buffer and 5 µL of lysate, corresponding to 6.25 x 10^5 HepG2 cells, was loaded per well on a 10% polyacrylamide gel. Proteins were transferred to a membrane and the membrane was blocked with 5% milk overnight at 4C. The membrane was probed with an alpha Fetoprotein monoclonal antibody (Product # MA1-19178) at a dilution of 1:500-1:2000 for 1 hour at RT, followed by a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:1000 for 1 hour at RT. Detection was performed using a chemiluminescent substrate

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Conditioned Media of Hep G2 (Lane 1) and HeLa (Lane 2). The blot was probed with anti-AFP Mouse Monoclonal Antibody (Product # MA1-19178, 1:1000 dilution) and detected by chemiluminescence using Goat anti Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 68 kDa band corresponding to AFP was detected in Hep G2 conditioned media but not in HeLa conditioned media which is negative for AFP expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of alpha Fetoprotein was performed on HepG2 whole cell lysates. Cell lysates were mixed with reducing (Lane 1) or non-reducing (Lane 2) Laemmli SDS-PAGE sample buffer and 5 µL of lysate, corresponding to 6.25 x 10^5 HepG2 cells, was loaded per well on a 10% polyacrylamide gel. Proteins were transferred to a membrane and the membrane was blocked with 5% milk overnight at 4C. The membrane was probed with an alpha Fetoprotein monoclonal antibody (Product # MA1-19178) at a dilution of 1:500-1:2000 for 1 hour at RT, followed by a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:1000 for 1 hour at RT. Detection was performed using a chemiluminescent substrate

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blotting analysis of human alpha-fetoprotein using mouse Monoclonal antibody AFP-01 (Product # MA1-19178) on lysates of HepG2 cell line and Jurkat cell line (negative control) under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse monoclonal antibody anti-alpha-fetoprotein followed by IRDye800-conjugated anti-mouse secondary antibody. Alpha-fetoprotein was detected at approximately 60kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of AFP was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with AFP Mouse Monoclonal Antibody (Product # MA1-19178) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic and Golgi localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

MA1-19178