Anti-NLRP3 antibody [RM1021] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling NLRP3 with ab283819 at 1/50 (11.4 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 increased mainly cytoplasmic staining in THP-1 cells treated with 12-O-Tetradecanoylphorbol-13-acetate (80 nM) for 16 h then replaced it with lipopolysaccharide (1 ug/ml) for 8 h. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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 (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) treated with 50nM PMA and 5ug/ml LPS for 24h(Red) / Untreated control (Green) cells labelling NLRP3 with ab283819 at 1/50 dilution (1ug) (Red) and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. NLRP3 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10ug with ab283819 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283819 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10 µg Lane 2 : ab283819 IP in THP-1 (Human monocytic leukemia monocyte) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283819 in THP-1 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lower bands should be cleavage fragment of NLRP3. (PMID : 26119741)

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/30 dilution

Lane 1:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate

Lane 2:

<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a> IP in THP-1 (Human monocytic leukemia monocyte) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/5000 dilution

false

Exposure time: 84s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 10ug/ml LPS for 8h(Red) / Untreated control (Green) cells labelling NLRP3 with ab283819 at 1/500 dilution (0.1ug) (Red) and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling NLRP3 with ab283819 at 1/50 (11.4 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 increased cytoplasmic staining in RAW 264.7 cells treated with lipopolysaccharide (10 ug/ml) for 8 h. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• IP

Supplier Data

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation. NLRP3 was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10ug with ab283819 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283819 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 µg Lane 2 : ab283819 IP in RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283819 in RAW 264.7 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lower bands should be cleavage fragment of NLRP3. (PMID : 26119741)

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/30 dilution

All lanes:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/5000 dilution

false

Exposure time: 84s

• WB

Supplier Data

Western blot - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed usingab283819, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-NLRP3 antibody (ab283819) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283819 was shown to bind specifically to NLRP3. Target of interest was observed at 118 kDa in untreated wild-type THP-1 cell lysates (lane 1) with no signal observed at this size in NLRP3 knockout cell line ab280063 (knockout cell lysate ab280122) (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

NLRP3 knockout THP-1 whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG H&L (800CW) at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 118 kDa,37 kDa

false

• WB

Supplier Data

Western blot - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed usingab283819, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : Jurkat (PMID : 19767079). Exposure time : Lane1-3 : 3min; Lane4-5 : 37 seconds; Lane6-7 : 10 seconds

All lanes:

Western blot - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

THP-1 treated with 80nM TPA overnight, then 1μg/ml lipopolysaccharide (LPS) for 8h whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 5:

RAW 264.7 treated with 1μg/ml lipopolysaccharide (LPS) for 6h whole cell lysate at 20 µg

Lane 6:

NR8383 (Rat lu macrophage (alveolar)) whole cell lysate at 20 µg

Lane 7:

NR8383 treated with 1μg/ml lipopolysaccharide (LPS) for 4h, then add 1μg/ml BFA for another 3h 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 1/100000 dilution

Observed band size: 118 kDa

false

• WB

Lab

Western blot - Anti-NLRP3 antibody [RM1021] - BSA and Azide free (AB314905)

This data was developed using ab283819, the same antibody clone in a different buffer formulation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

After extending the induction period to 16 hours, prolonged LPS stimulation may trigger cellular feedback regulation (PMID : 26412089), resulting in less pronounced induction effects. It is recommended to test varying time and concentration gradients to optimize induction.

All lanes:

Western blot - Anti-NLRP3 antibody [RM1021] (<a href='/en-us/products/primary-antibodies/nlrp3-antibody-rm1021-ab283819'>ab283819</a>) at 1/1000 dilution

Lanes 1, 3, 5 and 7:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 2h whole cell lysate at 20 µg

Lane 4:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 6h whole cell lysate at 20 µg

Lane 6:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 16h whole cell lysate at 20 µg

Lane 8:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 24h 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: 118 kDa

false

Exposure time: 3s