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Western blot coomassie blue stain

NLRP3 target tips

Related

  • Browse by Target

    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

    • Protein function, expression, and isoforms

    • Western Blot tips for working with NLRP3

    ​Sample preparation

    Electrophoresis

    Transferring

    Maximizing signal

    Recommended controls

    • References

    ​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)


    ​​


    ​

    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.

    Back to top


    Electrophoresis

    • Run samples in 8% or lower separating gel.

    ​​Back to top


    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.

    Back to top


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

    Back to top


    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

    Back to top


    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)

    View these tips as a PDF:

    • In English
    • In Mandarin




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