Anti-alpha 1 Fetoprotein antibody [RM1011]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

Negative control : No staining on human liver.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling alpha 1 Fetoprotein with ab284388 at 1/2000 (0.231 μg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab284388 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

Immunohistochemical analysis of paraffin-embedded Human hepatic carcinoma tissue labelling alpha 1 Fetoprotein with ab284388 at 1/2000 (0.231 μg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human hepatic carcinoma. The section was incubated with ab284388 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC

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Immunocytochemistry - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

This data was developed using ab284388, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling alpha 1 Fetoprotein with ab284388 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 (2μg/mL) dilution (Green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5μg/mL dilution (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing cytoplasmic staining in HepG2 cells is observed.

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2μg/mL) dilution.

• Flow Cyt

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Flow Cytometry - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell) cells labelling alpha 1 Fetoprotein with ab284388 at 1/500 dilution Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

alpha 1 Fetoprotein was immunoprecipitated from 0.35 mg HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10ug with ab284388 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab284388 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab284388 IP in HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab284388 in HepG2 whole cell lysate

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

Exposure time : 3 seconds

All lanes:

Immunoprecipitation - Anti-alpha 1 Fetoprotein antibody [RM1011] (ab284388)

Predicted band size: 68 kDa

Observed band size: 68 kDa

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• ICC

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Immunocytochemistry - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Hepa1-6 cells labelling alpha 1 Fetoprotein with ab284388 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2μg/mL dilution (Green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5μg/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing increased cytoplasmic staining in Hepa1-6 cells treated with Brefeldin A (300 ng/ml) for 24 h is observed.

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2μg/mL) dilution.

• Flow Cyt

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Flow Cytometry - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20 permeabilized Hepa1-6 (Mouse hepatoma epithelial cell) treated with 300ng/ml BFA for 24h (Right) / Untreated control (Left) cells labelling alpha 1 Fetoprotein with ab284388 at 1/500 dilution. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

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

Negative control : MDA-MB-231.

Exposure time : Lane 4-5 : 26 seconds ; Others : 5 seconds

All lanes:

Western blot - Anti-alpha 1 Fetoprotein antibody [RM1011] (ab284388) at 1/1000 dilution

Lane 1:

HepG2 (Human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Huh7 (Human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Hepa1-6 (Mouse hepatoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Hepa1-6 treated with 300ng/ml BFA for 24h whole cell lysate at 20 µg

Lane 6:

Mouse placenta lysate at 20 µg

Lane 7:

Rat placenta lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

• I-ELISA

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Indirect ELISA - Anti-alpha 1 Fetoprotein antibody [RM1011] (AB284388)

ELISA using ab284388 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.