Anti-NLRP3 antibody [EPR23094-1]

• WB

Supplier Data

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

Blocking and dilution buffer : 5% NFDM /TBST. NLRP3 is expressed at a much low level in normal tissues (PMID : 33538177, PMID : 25524927, PMID : 20688930, PMID : 35676979, PMID : 31923221, PMID : 24166187).

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

Rat spleen tissue lysate at 20 µg

Lane 2:

Rat thymus tissue lysate at 20 µg

Lane 3:

Rat lung tissue lysate at 20 µg

Lane 4:

Rat heart tissue lysate at 20 µg

Lane 5:

Rat kidney tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat hippocampus tissue lysate at 20 µg

Lane 8:

Rat spinal cord tissue lysate at 20 µg

Lane 9:

Rat placenta tissue lysate at 20 µg

Lane 10:

Rat ovary tissue lysate at 20 µg

Lane 11:

Rat liver 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 1/20000 dilution

false

Exposure time: 180s

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) cells labeling NLRP3 with ab263899 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

NLRP3 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate with ab263899 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263899 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : THP-1 whole cell lysate 10μg

Lane 2 : ab263899 IP in THP-1 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab263899 in THP-1 whole cell lysate

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

Predicted band size: 118 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

NLRP3 was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab263899 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263899 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : RAW 264.7 whole cell lysate 10μg

Lane 2 : ab263899 IP in RAW 264.7 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab263899 in RAW 264.7 whole cell lysate

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-NLRP3 antibody [EPR23094-1] (ab263899)

Predicted band size: 118 kDa

false

• WB

Lab

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

ab263899 was shown to react with NLRP3 in wild-type THP-1 cells in Western blot with loss of signal observed in a NLRP3 knockout cell line. Wild-type THP-1 and NLRP3 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab263899 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

Wild-type THP-1 lysate at 20 µg

Lane 2:

Wild-type THP-1 lysate treated with 200 ng/ml PMA, 48 hr at 20 µg

Lane 3:

NLRP3 knock-out THP-1 lysate at 20 µg

Lane 4:

NLRP3 knock-out THP-1 lysate treated with 200 ng/ml PMA, 48 hr at 20 µg

false

• WB

Lab

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

False colour image of Western blot : Anti-NLRP3 antibody [EPR23094-1] staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab263899 was shown to bind specifically to NLRP3. A band was observed at 118 kDa in wild-type THP-1 cell lysates with no signal observed at this size in NLRP3 knockout cell line ab280063 (knockout cell lysate ab280122). To generate this image, wild-type and NLRP3 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) at 1/20000 dilution.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/500 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

NLRP3 knockout THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human NLRP3 knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-nlrp3-knockout-thp-1-cell-line-ab280063'>ab280063</a>)

Predicted band size: 118 kDa

Observed band size: 118 kDa

false

• WB

Lab

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

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

Note : The band around 75kDa is the short form of NLRP3 (NLRP3s).

Low expression cell line : Jurkat (PMID : 19767079).

Exposure times : Lane 1 : 48 secs; Lane 2 : 3 min; Lanes 3-4 : 1 sec; Lane 5 : 6 secs.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

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

Lane 2:

Jurkat (human t cell leukemia t lymphocyte) whole cell lysate at 20 µg

Lane 3:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 4:

RAW 264.7 treated with 10 μg/ml LPS for 8 hours, whole cell lysate at 10 µg

Lane 5:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) 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

Predicted band size: 118 kDa

false

• WB

Collaborator

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

Blocking step : 2% SMP in PBS/T for 60 minutes at RT.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

Wild-type mouse BMDCs treated with 50 ng/ml LPS at 25000 Cells

Lane 2:

NLRP3 knockout mouse BMDCs treated with 50 ng/ml LPS at 25000 Cells

Lane 3:

Wild-type human THP1 cells treated with 200 ng/ml LPS at 25000 Cells

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/5000 dilution

Predicted band size: 118 kDa

true

Exposure time: 10s

This image is courtesy of Benedikt Saller, from Prof. Dr. Olaf Groß’ lab at the University of Freiburg.

• WB

Supplier Data

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

Blocking and Diluting buffer : 5% NFDM/TBST Lane 9 Exposure Time : 20 seconds NLRP3 is expressed at a much low level in normal tissues (PMID : 33538177, PMID : 25524927, PMID : 20688930, PMID : 35676979, PMID : 31923221, PMID : 24166187).

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

Mouse thymus tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse heart tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Mouse brain tissue lysate at 20 µg

Lane 6:

Mouse placenta tissue lysate at 20 µg

Lane 7:

Mouse ovary tissue lysate at 20 µg

Lane 8:

Mouse liver tissue lysate at 20 µg

Lane 9:

Raw 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage) 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

Predicted band size: 118 kDa

Observed band size: 118 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

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

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

All lanes:

Rat spleen 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

Predicted band size: 118 kDa

false

Exposure time: 3min

• I-ELISA

Unknown

Indirect ELISA - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

ELISA analysis of NLRP3 recombinant protein at 1000 ng/mL with ab263899. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

• WB

Unknown

Western blot - Anti-NLRP3 antibody [EPR23094-1] (AB263899)

Stimulation is required to allow detection of the NLRP3 protein in C6, PC-12 and NIH/3T3 cell lines.

All lanes:

Western blot - Anti-NLRP3 antibody [EPR23094-1] (ab263899) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) 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 1/10000 dilution

Predicted band size: 118 kDa

Observed band size: 118 kDa

false

Exposure time: 15s