Western blot coomassie blue stain

NLRP3 target tips

NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) is a protein encoded by the NLRP3 gene. NLRP3 is expressed predominantly in macrophages and plays a crucial role in innate immunity and inflammation.

​Overview

​Sample preparation

Electrophoresis

Transferring

Maximizing signal

Recommended controls

​Protein function, expression, and isoforms

Protein function 

  • NLRP3 may function as an inducer of apoptosis.
  • It interacts selectively with ASC, and this complex may function as an upstream activator of NF-kappa-B signaling. 
  • It inhibits TNF-alpha-induced activation & nuclear translocation of RELA/NF-KB p65, and also inhibits transcriptional activity of RELA. 
  • NLRP3 activates caspase-1 in response to various triggers including bacterial or viral infection, which leads to the processing and release of IL1B and IL18.

Expression

  • NLRP3 is expressed in blood leukocytes and is also strongly expressed in polymorphonuclear cells and osteoblasts
  • NLRP3 is undetectable or expressed at a lower magnitude in B- and T-lymphoblasts respectively 
  • High levels of NLRP3 expression are detected in chondrocytes
  • NLRP3 is also detected in non-keratinizing epithelia of the oropharynx, esophagus, and ectocervix, and in the urothelial layer of the bladder

Isoforms

  • Human: Isoform 1: 84 kD (predicted)
  • Human: Isoform 2: 106 kD (predicted)
  • Human: Isoform 3-6: 110-120 kD (predicted)
  • Mouse: Isoform 1: 96 kD (predicted)
  • Mouse: Isoform 2-4: 110-120 kD (predicted)
  • Rat: Isoform 1: 119 kD (predicted)


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Western blot tips

NLRP3 is regulated by post-translational modifications, such as ubiquitination and phosphorylation, and has several isoforms. Therefore, several bands may exist in a western blot assay. The predicted molecular weight of NLRP3 is about 118 kDa.

Sample preparation
  • Add protease inhibitors to avoid protein degradation.
  • Ultrasonicate cell samples to isolate more protein
  • Keep sample on ice throughout the process
  • Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.

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Electrophoresis

  • Run samples in 8% or lower separating gel.

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Transferring

  • We recommend adding SDS to a final concentration of 0.1% in the transfer buffer.
  • Wash PVDF membrane to remove methanol completely.
  • Determine whether the transfer is successful by visualization of proteins in membranes using Ponceau S.

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Maximizing signal
  • The level of NLRP3 expression can be elevated when cells are treated with lipopolysaccharide (LPS) 1.​

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Recommended controls


Positive:
Human THP1 WT cells treated with 200 ng/ml LPS for 3h;
Rat spleen lysate


Negative:
Jurkat (human t cell leukemia t lymphocyte) whole cell lysate

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Find full information on working with NLRP3: 
In English (link to pdf) 
In Mandarin (Link to pdf) 

References

  1. Qiao, Y., Wang, P., Qi, J., Zhang, L., Gao, C. TLR-induced NF-κB activation regulates NLRP3 expression in murine macrophages. FEBS Lett. 586, 1022-6 (2012)

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