Anti-AACT antibody [EPR17088-68]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-AACT antibody [EPR17088-68] (AB205198)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (Human Burkitt's lymphoma cell line) cells labeling AACT with ab205198 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and weak nuclear staining on Raji cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab205198 at 1/50 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AACT antibody [EPR17088-68] (AB205198)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling AACT with ab205198 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab205198) at 1/500 dilution. Cytoplasmic staining on human tonsil tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-AACT antibody [EPR17088-68] (AB205198)

AACT was immunoprecipitated from 1 mg of Human plasma lysate with ab205198 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab205198 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.

Lane 1 : Human plasma lysate, 10 μg (Input).

Lane 2 : ab205198 IP in Human plasma lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab205198 in Human plasma lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-AACT antibody [EPR17088-68] (ab205198)

Predicted band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-AACT antibody [EPR17088-68] (AB205198)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-AACT antibody [EPR17088-68] (ab205198) at 1/1000 dilution

All lanes:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 47 kDa

Observed band size: 50-70 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-AACT antibody [EPR17088-68] (AB205198)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-AACT antibody [EPR17088-68] (ab205198) at 1/1000 dilution

All lanes:

Human blood 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 1/50000 dilution

Predicted band size: 47 kDa

Observed band size: 50-70 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-AACT antibody [EPR17088-68] (AB205198)

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID : 9880565).

All lanes:

Western blot - Anti-AACT antibody [EPR17088-68] (ab205198) at 1/2000 dilution

All lanes:

Human plasma at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 47 kDa

Observed band size: 50-70 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-AACT antibody [EPR17088-68] (AB205198)

Blocking/Dilution buffer : 5% NFDM/TBST.

De-glycosylation treatment with PNGase, demonstrates a molecular weight shift from 50-70kDa to 46kDa. This result is consistent with what has been described in literature the literature (PMID : ) 9880565.

All lanes:

Western blot - Anti-AACT antibody [EPR17088-68] (ab205198) at 1/2000 dilution

Lane 1:

Untreated Human plasma at 10 µg

Lane 2:

Human plasma treated with PNGase F at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 50-70 kDa

false

Exposure time: 4s

• WB

Lab

Western blot - Anti-AACT antibody [EPR17088-68] (AB205198)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

All lanes:

Western blot - Anti-AACT antibody [EPR17088-68] (ab205198) at 1/1000 dilution

All lanes:

His-Tagged Human SERPINA3 (aa 24 to 324) recombinant protein 100ng

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

Predicted band size: 47 kDa

Observed band size: 33 kDa

false

Exposure time: 3min