Anti-FGL1 antibody [EPR24018-27]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling FGL1 with ab275091 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human liver (PMID : 11470158). The section was incubated with ab275091 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell) cells labelling FGL1 with ab275091 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling FGL1 with ab275091 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control : No staining in human cerebrum (PMID : 14981537). The section was incubated with ab275091 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling FGL1 with ab275091 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human hepatocellular carcinoma (PMID : 14981537). The section was incubated with ab275091 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• IP

Unknown

Immunoprecipitation - Anti-FGL1 antibody [EPR24018-27] (AB275091)

FGL1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab275091 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275091 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 10 ug

Lane 2 : ab275091 IP in HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of abID1 in HepG2 whole cell lysate

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-FGL1 antibody [EPR24018-27] (ab275091)

Predicted band size: 36 kDa

Observed band size: 34 kDa

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• WB

Lab

Western blot - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Western blot : Rabbit Monoclonal [EPR24018-27] to FGL1 ab275091 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 36 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween 20 (TBS-T) before incubation with primary antibodies overnight at 4 C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-FGL1 antibody [EPR24018-27] (ab275091) at 1/1000 dilution

Lane 1:

Wild-type A549 IL-6 (5 ng/mL, 48 h) + BFA (5 g/mL, last 6 h) at 20 µg

Lane 2:

Wild-type A549 IL-6 (0 ng/mL, 48 h) + BFA (5 g/mL, last 6 h) at 20 µg

Lane 3:

Wild-type A549 BFA (0 g/mL, 6 h) at 20 µg

Lane 4:

FGL1 knockout A549 IL-6 (5 ng/mL, 48 h) + BFA (5 g/mL, last 6 h) at 20 µg

Lane 5:

FGL1 knockout A549 IL-6 (0 ng/mL, 48 h) + BFA (5 g/mL, last 6 h) at 20 µg

Lane 6:

FGL1 knockout A549 BFA (0 g/mL, 6 h) at 20 µg

Lane 7:

HepG2 at 20 µg

Lane 8:

HeLa at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 36 kDa

Observed band size: 36 kDa

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• WB

Unknown

Western blot - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 15 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID : 11004478).

Negative control : Human brain (PMID : 11470158).

All lanes:

Western blot - Anti-FGL1 antibody [EPR24018-27] (ab275091) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human brain tissue lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 36 kDa

Observed band size: 34 kDa

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• WB

Unknown

Western blot - Anti-FGL1 antibody [EPR24018-27] (AB275091)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-FGL1 antibody [EPR24018-27] (ab275091) at 1/1000 dilution

All lanes:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 36 kDa

Observed band size: 34 kDa

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