重组Anti-IFNGR1抗体[EPR7866] - Low endotoxin，Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IFNGR1 (red) with ab134070 at a 1/1000 dilution. Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134070).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling IFNGR1 with ab134070 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134070).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling IFNGR1 with purified ab134070 at 1/100 dilution (10 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL) was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134070).

• WB

Lab

Western blot - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

This data was developed using the same antibody clone in a different buffer formulation (ab134070).

Lanes 1 - 4 : Merged signal (red and green). Green - ab134070 observed at 60-80 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab134070 was shown to react with IFNGR1 in wild-type HEK-293 cells in western blot with loss of signal observed in IFNGR1 knockout sample. Wild-type and IFNGR1 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab134070 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IFNGR1 antibody [EPR7866] (<a href='/products/primary-antibodies/ifngr1-antibody-epr7866-ab134070'>ab134070</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

IFNGR1 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human IFNGR1 knockout HEK-293 cell line (<a href='/products/cell-lines/human-ifngr1-knockout-hek-293-cell-line-ab269471'>ab269471</a>)

Lane 3:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 54 kDa

Observed band size: 60-80 kDa

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• WB

Lab

Western blot - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

This data was developed using the same antibody clone in a different buffer formulation (ab134070 ).

Western blot : Anti-IFNGR1 antibody [EPR7866] (ab134070) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab134070 was shown to bind specifically to IFNGR1. A band was observed at 70-75 kDa in wild-type A549 cell lysates with no signal observed at this size in IFNGR1 knockout cell line. To generate this image, wild-type and IFNGR1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IFNGR1 antibody [EPR7866] (<a href='/products/primary-antibodies/ifngr1-antibody-epr7866-ab134070'>ab134070</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

IFNGR1 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human IFNGR1 knockout A549 cell line (<a href='/products/cell-lines/human-ifngr1-knockout-a549-cell-line-ab287503'>ab287503</a>)

Lane 3:

Wild-type HEK-293 ab259780 cell lysate at 20 µg

Lane 4:

IGNGR1 knockout HEK-293 <a href='/products/cell-lines/human-ifngr1-knockout-hek-293-cell-line-ab269471'>ab269471</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 70-75 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-IFNGR1 antibody [EPR7866] - Low endotoxin, Azide free (AB246696)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.