Anti-NLRP3 antibody [EPR23073-96] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NLRP3 antibody [EPR23073-96] - BSA and Azide free (AB272702)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (untreated)(-) and RAW 264.7 (+LPS 10ug/ml 8h)(+) cells labelling NLRP3 with ab270449 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in RAW 264.7 after treatment with LPS. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab270449).

• WB

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Western blot - Anti-NLRP3 antibody [EPR23073-96] - BSA and Azide free (AB272702)

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

The molecular weight/expression profile observed is consistent with what has been described in the literature (PMID : 26939933, 30315268 ).

The band around 75kDa corresponds to the short form of NLRP3.

Exposure time : 54 seconds.

This data was develoed using the same antibody clone in a different buffer formulation containing PBS, glycerol, BSA and sodium azide (ab270449).

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23073-96] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-epr23073-96-ab270449'>ab270449</a>) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 10 μg/ml Lipopolysaccharides (LPS) for 8 hours whole cell lysate at 20 µg

Lane 3:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 118 kDa

Observed band size: 118 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-NLRP3 antibody [EPR23073-96] - BSA and Azide free (AB272702)

NLRP3 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) (treated with 10 ug/ml Lipopolysaccharides (LPS) for 8 hours) whole cell lysate with ab270449 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270449 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 treated with 10 ug/ml Lipopolysaccharides (LPS) for 8 hours, whole cell lysate 10 ug

Lane 2 : ab270449 IP in RAW 264.7 treated with 10 ug/ml Lipopolysaccharides(LPS) for 8 hours, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270449 in RAW 264.7 treated with 10 ug/ml Lipopolysaccharides(LPS) for 8 hours, whole cell lysate

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

Exposure time : 24 seconds.

The band around 75kDa corresponds to the short form of NLRP3.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab270449).

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [EPR23073-96] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-epr23073-96-ab270449'>ab270449</a>)

Predicted band size: 118 kDa

Observed band size: 118 kDa

false