Anti-Interferon gamma antibody [RM1245]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Interferon gamma antibody [RM1245] (AB322926)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) cells labelling Interferon gamma with ab322926 at 1/50 (9.98 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic and membranous staining in No-GFP-CD16.NK-92 cell line treated with Phorbol-12-myristate-13-acetate (80nM) and Ionomycin(3uM) for 5 hours, then add Brefeldin A (300ng/ml) for 4 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt

Lab

Flow Cytometry - Anti-Interferon gamma antibody [RM1245] (AB322926)

Flow cytometric analysis of 0.1% Tween-20 permeabilized, 2% paraformaldehyde-fixed Human PBMC (human peripheral blood mononuclear cell). Cell stimulation was performed with cell stimulation cocktail for 6 hours (bottom) compared to unstimulated cells (top). Interferon gamma was labeled with ab322926 at 1/2500 dilution compared with an Isotype control, Rabbit monoclonal IgG (ab172730) (left).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Human PBMC were co-stained with CD4 conjugated to Alexa Fluor^®647

• Flow Cyt

Lab

Flow Cytometry - Anti-Interferon gamma antibody [RM1245] (AB322926)

Flow cytometric analysis of 0.1% Tween-20 permeabilized, 2% paraformaldehyde-fixed Human PBMC (human peripheral blood mononuclear cell). Cell stimulation was performed with cell stimulation cocktail for 6 hours (bottom) compared to unstimulated cells (top). Interferon gamma was labeled with ab322926 at 1/2500 dilution compared with an Isotype control, Rabbit monoclonal IgG (ab172730) (left).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Human PBMC were co-stained with CD19 conjugated to PE/Cy7.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Interferon gamma antibody [RM1245] (AB322926)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) stimulated with 80nM TPA and 3uM Ionomycin for 5 hours then add 300ng/mL BFA for 4 hours(Upper left and right) / No-GFP-CD16.NK-92 unstimulated cell (Lower left and right) cells labelling Interferon gamma with ab322926 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-Interferon gamma antibody [RM1245] (AB322926)

Interferon gamma was immunoprecipitated from 0.35 mg No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (ab120297) and 3uM Ionomycin (ab120116) for 5 hours, 300ng/ml BFA was added for last 4 hours whole cell lysate with ab322926 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322926 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (ab120297) and 3uM Ionomycin (ab120116) for 5 hours, 300ng/ml BFA was added for last 4 hours whole cell lysate
Lane 2 : ab322926 IP in No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (ab120297) and 3uM Ionomycin (ab120116) for 5 hours, 300ng/ml BFA was added for last 4 hours whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322926 in No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (ab120297) and 3uM Ionomycin (ab120116) for 5 hours, 300ng/ml BFA was added for last 4 hours whole cell lysate

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

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

All lanes:

Immunoprecipitation - Anti-Interferon gamma antibody [RM1245] (ab322926) at 1/30 dilution

All lanes:

No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) and 3uM Ionomycin (<a href='/en-us/products/biochemicals/ionomycin-ca2-salt-ca2-ionophore-ab120116'>ab120116</a>) for 5 hours, 300ng/ml BFA was added for last 4 hours 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

false

Exposure time: 111s

• WB

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Western blot - Anti-Interferon gamma antibody [RM1245] (AB322926)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23129404 and PMID : 6423641).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Interferon gamma antibody [RM1245] (ab322926) at 1/1000 dilution

Lane 1:

Untreated No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate at 20 µg

Lane 2:

No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) and 3uM Ionomycin (<a href='/en-us/products/biochemicals/ionomycin-ca2-salt-ca2-ionophore-ab120116'>ab120116</a>) for 5 hours, 300 ng/ml BFA was added for last 4 hours 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 1/20000 dilution

Observed band size: 17 kDa,19 kDa,23 kDa,36 kDa

false

Exposure time: 92s

• I-ELISA

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Indirect ELISA - Anti-Interferon gamma antibody [RM1245] (AB322926)

Indirect ELISA analysis of ab322926 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human IFNG.

Antigen concentration : 1000 ng/ml