Anti-GBP3 antibody [EPR30342-524]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GBP3 antibody [EPR30342-524] (AB326333)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) and J774A.1 treated with IFN gamma(mouse)(200ng/ml) for 24hours and Lipopolysaccharides(50ng/ml) for 24 hours cells labelling GBP3 with ab326333 at 1/50 (9.9 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in J774A.1 treated with IFN gamma(mouse)(200ng/ml) for 24hours and Lipopolysaccharides(50ng/ml) for 24hours (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).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326333 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• WB

Lab

Western blot - Anti-GBP3 antibody [EPR30342-524] (AB326333)

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

The bands beneath the target band (70 kDa) in lanes 2 and 3 are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-GBP3 antibody [EPR30342-524] (ab326333) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse lymph node tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue 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

Observed band size: 70 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-GBP3 antibody [EPR30342-524] (AB326333)

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

The band beneath the target band (70 kDa) in lanes 2 and 4 is likely to be degraded target fragment.

The expression of GBP3 is upregulated in response to IFN-gamma( PMID : 9659399)

To minimize protein degradation, cells(Lanes 5-8) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-GBP3 antibody [EPR30342-524] (ab326333) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 treated with 100ng/ml IFN gamma (mouse) for 48 hours whole cell lysate at 10 µg

Lane 3:

Untreated J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 10 µg

Lane 4:

J774A.1 treated with 200ng/ml IFN gamma (mouse) and 50ng/ml LPS for 24 hours whole cell lysate at 10 µg

Lane 5:

Untreated NIH/3T3 (mouse embryonic fibroblast) fresh whole cell lysate at 10 µg

Lane 6:

NIH/3T3 treated with 100ng/ml IFN gamma (mouse) for 48 hours fresh whole cell lysate at 10 µg

Lane 7:

Untreated J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) fresh whole cell lysate at 10 µg

Lane 8:

J774A.1 treated with 200ng/ml IFN gamma (mouse) and 50ng/ml LPS for 24 hours fresh whole cell lysate at 10 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

Observed band size: 70 kDa,36 kDa

false

Exposure time: 26s