Anti-HERV-FRD antibody [EPR30312-632]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling HERV-FRD with ab326102 at 1/50 (9.94 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Cytoplasmic staining in human placenta (PMID : 18215254; PMID : 18988732).

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized JEG-3 (human placenta epithelial cell) HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HERV-FRD with ab326102 at 1/50 (9.94 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing membranous staining in JEG-3 cell line and no staining in HeLa cell line(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Negative control : HeLa. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326102 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

HERV-FRD was immunoprecipitated from 0.35 mg JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate with ab326102 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326102 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate
Lane 2 : ab326102 IP in JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326102 in JEG-3 whole cell lysate

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

Exposure time : 26 seconds.

All lanes:

Immunoprecipitation - Anti-HERV-FRD antibody [EPR30312-632] (ab326102) at 1/1000 dilution

Lane 1:

JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate at 10 µg

Lane 2:

ab326102 at 1/30 IP in JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab326102 in JEG-3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 59 kDa,70 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

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

Low expression : kidney, liver, small intestine (PMID : 10693809)

The molecular weight observed is consistent with what has been described in the literature (PMID : 35596004; PMID : 18650494).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HERV-FRD antibody [EPR30312-632] (ab326102) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

human liver tissue lysate at 20 µg

Lane 3:

Human small intestine tissue lysate at 20 µg

Lane 4:

human placenta tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 59 kDa,70 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

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

Negative control : HeLa

The molecular weight observed is consistent with what has been described in the literature (PMID : 35596004; PMID : 18650494).

HERV-FRD is a glycoprotein of approximately 59 and 70 kDa and is detected as 37 kDa and 50 kDa band after treatment with the deglycosylation enzyme Peptide : N-glycosidase F (PNGase F)(Lane 4).

Lanes 3-4 of this blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HERV-FRD antibody [EPR30312-632] (ab326102) at 1/1000 dilution

Lane 1:

JEG-3 (human placenta epithelial cell) whole cell lysate at 48 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 48 µg

Lane 3:

Untreated JEG-3 (human placenta epithelial cell) whole cell lysate at 48 µg

Lane 4:

JEG-3 lysate treated with Protein Deglycosylation Peptide:N-glycosidase F (PNGase F) at 48 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: 59 kDa,70 kDa,36 kDa

true

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HERV-FRD antibody [EPR30312-632] (AB326102)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling HERV-FRD with ab326102 at 1/50 (9.94 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : Negative staining in human kidney (PMID : 10693809).

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins