Figure 1: NLRP3 protein structure.

NLRP3 Introduction

Protein Function

• NLRP3 functions as a sensor of inflammasomes, playing a crucial role in innate immunity and inflammation.
• NLRP3 selectively interacts with ASC to form a complex, which acts as an upstream activator of the NF-kappa-B signaling pathway.
• NLRP3 also inhibits the transcriptional activity of RELA.
• NLRP3 inhibits TNF-alpha-induced activation and nuclear translocation of RELA/NF-KB p65.
• NLRP3 activates caspase-1 in response to a number of triggers including bacterial or viral infection which leads to processing and release of IL1B and IL18.

Protein Characteristics

• There are 6 isoforms of NLRP3 in humans and 4 isoforms in mice, all of which can be detected.
• NLRP3 is a low-expressed protein, and detection may be facilitated by LPS stimulation.

Protein Expression

• Expressed in blood leukocytes. Strongly expressed in polymorphonuclear cells and osteoblasts.
• Undetectable or expressed at a lower magnitude in B- and T-lymphoblasts, respectively.
• High level of expression detected in chondrocytes.
• Expression detected in non-keratinizing epithelium of the oral, esophageal and ectocervical mucosa, and in the stratified epithelium covering the bladder and ureter.

Protein Localization

• Cytoplasm

Figure 2: ICC Experimental Results of NLRP3 Protein. Antibody Used: Anti-NLRP3 (product ab270449). Green: NLRP3; Red: alpha Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

• Human (Q96P20):

  • Isoform 3 (Q96P20-3): 84 kDa (predicted)
  • Isoform 1 (Q96P20-2): 106 kDa (predicted)
  • Isoforms 2, 4-6 (Q96P20-1, Q96P20-4~ Q96P20-6): 111-118 kDa (predicted)

• Mouse (Q8R4B8):

  • Isoform 4 (Q8R4B8-4): 96 kDa (predicted)
  • Isoforms 1-3 (Q8R4B8-1~ Q8R4B8-3): 112-118 kDa (predicted)

• Rat (D4A523):
  • 119 kDa (predicted)
• Ubiquitination

WB Experiment Tips

Precautions

• NLRP3 is expressed at low levels in normal tissues such as mouse liver and kidney. Prior to detection, it is necessary to confirm the expression level of the target protein. It is also recommended to conduct experiments with a positive control recommended in the antibody product instructions.
• For certain cells like RAW264.7, LPS treatment can increase intracellular NLRP3 expression. Therefore, LPS stimulation is advantageous for resolving issues such as no signal or weak signal in WB experiments. (Refer to positive controls for guidance.)
• In WB experiments, NLRP3 may exhibit multiple bands due to the presence of multiple isoforms in humans and mice.
• Due to the various modifications of NLRP3, the observed band sizes in WB may not completely match the predicted molecular weights.

Positive Controls:

• Human: THP-1 (human monocytic leukemia monocyte) whole cell lysate.
• Mouse: RAW 264.7 whole cell lysate treated with 10 µg/ml lipopolysaccharide (LPS) for 8 hours.
• Rat: Spleen tissue lysate.

Negative Controls (No or Low Expression):

• Human: Jurkat whole cell lysate.
• Mouse: Liver, kidney, heart tissue lysates, NIH/3T3 whole cell lysate.
• Rat: C6 (Rat glial tumor glial cell), PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates.

Example Results

Figure 3: WB - Anti-NLRP3 antibody [EPR23073-96] (ab263899)

Lane 1: Mouse thymus tissue lysate
Lane 2: Mouse lung tissue lysate
Lane 3: Mouse heart tissue lysate
Lane 4: Mouse kidney tissue lysate
Lane 5: Mouse brain tissue lysate
Lane 6: Mouse placenta tissue lysate
Lane 7: Mouse ovary tissue lysate
Lane 8: Mouse liver tissue lysate
Lane 9: Raw264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Loading amount: 20 µg per lane
Predicted band size: 118 kDa
Observed band size: 118 kDa

Exposure time: Lanes 1-8: 180 seconds; Lane 9: 20 seconds

Figure 4: WB - Anti-NLRP3 antibody [EPR23073-96] (ab263899)

Lane 1: THP-1 (human monocytic leukemia monocyte) whole cell lysate, 20 µg
Lane 2: Jurkat (human T cell leukemia T lymphocyte) whole cell lysate, 20 µg
Lane 3: Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate, 10 µg
Lane 4: RAW 264.7 whole cell lysate treated with 10 µg/ml LPS for 8 hours, 10 µg
Lane 5: J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate, 20 µg

Predicted band size: 118 kDa
Note: Bands around 75 kDa are NLRP3 isoforms.

Exposure time: Lane 1: 48 seconds; Lane 2: 3 minutes; Lanes 3-4: 1 second; Lane 5: 6 seconds

Figure 5: WB - Anti-NLRP3 antibody [EPR23094-1] - BSA and Azide free (ab264468).

Lane 1: C6 (Rat glial tumor glial cell) whole cell lysate
Lane 2: RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3: PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate

Loading: 10 µg per lane
Predicted band size: 118 kDa
Observed band size: 118 kDa

Exposure time: 15 seconds

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add inhibitor cocktail to prevent degradation of target proteins.
2. Keep the samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford assay, Lowry assay, or BCA assay.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.
2. For target proteins with larger molecular weights (e.g., >100 kDa), it is recommended to use an 8% separating gel for electrophoresis.

Transfer:

1. Add SDS to the transfer buffer to a final concentration of 0.1%.
2. After activating the PVDF membrane, thoroughly wash it to completely remove any residual methanol.
3. It is recommended to use 10% methanol in the transfer buffer.
4. It is suggested to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Reference

1. Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 inflammasome: an overview of mechanisms of activation and regulation. Int J Mol Sci. (2019). 20(13):3328. doi: 10.3390/ijms20133328.
2. Nakanishi H, Kawashima Y, Kurima K et al. NLRP3 mutation and cochlear autoinflammation cause syndromic and nonsyndromic hearing loss DFNA34 responsive to anakinra therapy. Proc Natl Acad Sci U S A. (2017).114(37): E7766-E7775. doi: 10.1073/pnas.1702946114.