All lanes : Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : FCGRT knockout A549 cell lysate
Lane 3 : HEK293 cell lysate
Lane 4 : Lncap cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 40 kDa
Observed band size: 40 kDa

Lanes 1-4: Merged signal (red and green). Green - ab228975 observed at 40 kDa. Red - loading control ab7291 observed at 50 kDa.

ab228975 Anti-FCGRT/FCRN antibody [EPR22627-25] was shown to specifically react with FCGRT/FCRN in wild-type A549 cells. Loss of signal was observed when knockout cell line ab265075 (knockout cell lysate ab257435) was used. Wild-type and FCGRT/FCRN knockout samples were subjected to SDS-PAGE. ab228975 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling FCGRT/FCRN with ab228975 at 1/4000 dilution  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human liver (PMID: 28402755) is observed. The section was incubated with ab228975 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

All lanes : Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1 : Human placenta tissue lysate
Lane 2 : Human placenta treated with PNGase F tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 40 kDa


Exposure time: 3 minutes

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

Human FCGRT/FCRN possesses one potential glycosylation site. The molecular weight of deglycosylated FCGRT/FCRN is consistent with the literature (PMID: 12163559).

All lanes : Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate
Lane 3 : LNCaP (human prostate carcinoma epithelial cell), whole cell lysate
Lane 4 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa

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

Exposure times: Lane 1: 37 secs; Lane 2-4: 3 mins.

Negative control: LNCaP (PMID: 27974681) and HeLa (PMID: 21216798).

FCGRT/FCRN was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab228975 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab228975 1/1000 dilution (0.55 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2: ab228975  IP in HEK-293 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab228975 in HEK-293 whole cell lysate.

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

Exposure time: 100 seconds.

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling FCGRT/FCRN with ab228975 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human spleen, arrows indicate the macrophages (PMID: 28402755) is observed .The section was incubated with ab228975 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling FCGRT/FCRN with ab228975 at 1/4000 dilution (0.14 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on endothelial cells of human spleen (PMID: 28402755) is observed. The section was incubated with ab228975 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Flow cytometric analysis of HeLa (human cervix adenocarcinoma epithelial cell, Left) / THP-1 (human monocytic leukemia monocyte, Right) cells labeling FCGRT/FCRN with ab228975 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 647, ab150079) at 1/2000 dilution was used as the secondary antibody. Bright fluorophore (like AF647) conjugated secondary antibodies are recommended to get good separation.

Negative control: HeLa (PMID: 21216798).

Gated on viable cells.