Anti-FCGRT/FCRN antibody [EPR22627-25]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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).

• Flow Cyt

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Flow Cytometry - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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).

• IP

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Immunoprecipitation - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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.

All lanes:

Immunoprecipitation - Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975)

Predicted band size: 40 kDa

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• WB

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Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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:

Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1:

Human placenta tissue lysate at 20 µg

Lane 2:

Human placenta treated with PNGase F tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 40 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

Lanes 1-4 : Merged signal (red and green). Green - ab228975 observed at 40 kDa. Red - loading control ab8245 observed at 38 kDa.

ab228975 Anti-FCGRT/FCRN antibody [EPR22627-25] was shown to specifically react with FCGRT/FCRN in wild-type HEK-293T 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-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

All lanes:

Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (non-ATCC) cell lysate at 20 µg

Lane 2:

Western blot - Human FCGRT (FCRN) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-fcgrt-fcrn-knockout-hek-293t-cell-lysate-ab257435'>ab257435</a>)

Lane 2:

Human FCGRT (FCRN) knockout HEK-293T (non-ATCC) cell lysate (<a href='/en-us/products/cell-lysates/human-fcgrt-fcrn-knockout-hek-293t-cell-lysate-ab257435'>ab257435</a>)

Lane 3:

HEK293 (ATCC) cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 40 kDa

Observed band size: 40 kDa

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• WB

Unknown

Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (AB228975)

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).

All lanes:

Western blot - Anti-FCGRT/FCRN antibody [EPR22627-25] (ab228975) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 3:

LNCaP (human prostate carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 40 kDa

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