I want to use NLRP3 to stimulate a human cell line to check whether there is expression of IL-1beta or IL-18. Should I choose the partial or full-length NLRP3 protein? Do you have recommended dosage of NLRP3 for cell culture? Thanks.

This product is produced by a Taiwanese company called Abnova and we distribute it for them. They do not test the activity of their recombinant proteins. The proteins are produced in a cell-free wheat germ system with an N-terminal 26kDa GST-tag. Due to this expression system, in our experience, the proteins do not always undergo the same folding and post-transnational modifications they might undergo in an endogenous cell. Because of this, we do not recommend this protein for use in functional assays or cell culture and do not guarantee them for this use. They are mainly intended for use in Western Blots as a positive control with their antibody.

I'm using the Nalp3 recombinant protein as a positive control in a Western Blot. How much protein do you suggest I load onto the gel?

Abnova, the manufacturer of H00114548-P01, suggests loading 0.1-0.2ug per lane of purified protein in Western blot.

Recombinant Human NLRP3/NALP3 GST (N-Term) Protein

Name: Recombinant Human NLRP3/NALP3 GST (N-Term) Protein
Brand: Novus Biologicals
SKU: H00114548-P01

Novus Biologicals, part of Bio-Techne | Catalog # H00114548-P01

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Product Datasheet / COA / SDS

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Product Specifications
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Source

Wheat germ

Tag

GST (N-Term)

Conjugate

Unconjugated

Applications

ELISA, Affinity Purification, Microarray, SDS-PAGE, Western Blot

View all NLRP3/NALP3 Proteins and Enzymes »

Product Specifications

Description

A recombinant protein with GST tag at N-terminal corresponding to the amino acids 1-1036 of Human NLRP3

Source: Wheat Germ (in vitro)

Amino Acid Sequence: MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMIDFNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGLLEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCTGLKQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTRMDHMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGLVNSHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRCGLSHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLTSACCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVCCSALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWDLSTLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALETLQEEKPELTVVFEPSW

Purity

>80% by SDS-PAGE and Coomassie blue staining

Predicted Molecular Mass

144.6 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Activity

This protein was produced in an in vitro wheat germ expression system that should preserve correct conformational folding that is necessary for biological function. While it is possible that this protein could display some level of activity, the functionality of this protein has not been explicitly measured or validated.

Application Notes

Use in functional assay reported in scientific literature (PMIDs: 23871209 & 29531021)

Protein / Peptide Type

Recombinant Protein

Scientific Data Images for Recombinant Human NLRP3/NALP3 GST (N-Term) Protein

SDS-PAGE: Recombinant Human NLRP3/NALP3 GST (N-Term) Protein [H00114548-P01]

SDS-Page: Recombinant Human NLRP3/NALP3 Protein [H00114548-P01] - 12.5% SDS-PAGE stained with Coomassie Blue.

Formulation, Preparation and Storage

H00114548-P01

Preparation Method	in vitro wheat germ expression system
Formulation	50 mM Tris-HCl, 10 mM reduced Glutathione, pH 8.0 in the elution buffer.
Preservative	No Preservative
Concentration	Please see the vial label for concentration. If unlisted please contact technical services.
Shipping	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage	Store at -80C. Avoid freeze-thaw cycles.

Background: NLRP3/NALP3

NACHT, LRR and PYD domains-containing protein 3 (NALP3), Nucleotide-Binding Oligomerization Domain, or Leucine Rich Repeat and Pyrin Domain Containing 3 (NLRP3) (human NLRP3-isoform 2 theoretical molecular weight 118kDa) acts as a cytosolic receptor for stress-signals initiated by a wide variety of pathogen-associated molecular patterns (PAMPS) and danger-associated molecular patterns (DAMPs) (1). NLRP3 belongs to the family of NOD-like receptors, a type of pattern-recognition receptor (PRR) which participates in innate immune responses. The exact mechanisms leading to NLRP3 activation are still not fully resolved. Some proposed mechanisms for NLRP3 inflammasome activation include induced ion channel flux as exemplified by potassium efflux, sodium influx and calcium signaling, generation of mitochondrial reactive oxygen species (ROS), and lysosomal destabilization (2). Activation of NLRP3 induces its association with the adaptor protein Apoptosis-associated Speck-like protein containing CARD (ASC) forming a complex that binds to Caspase-1 (1, 2). The inflammasome complex, comprised of NLRP3, ASC and Caspase 1, induces the processing of pro-IL-1beta and pro-IL-18 and release of the mature inflammatory cytokines IL-1beta and IL-18 and pyroptosis (1-3).

NLRP3 is expressed in a variety of cell types such as monocytes, dendritic cells, lymphocytes and epithelial cells (3). Abnormal NLRP3 activation may occur as the result of inherited mutations and is associated with diseases such as hereditary periodic fevers (HPFs) and familial cold autoinflammatory syndrome (FCAS). Additionally, abnormal NLRP3 activation is associated with a variety of diseases such as gout, obesity, atherosclerosis, Alzheimers, multiple sclerosis and type 2 diabetes (1, 3). NLRP3 inflammasome is regulated by several post-translational modifications (e.g., ubiquitination, phosphorylation and sumoylation) as well as by various interacting proteins (e.g., JNK1, FBXO3, TXNIP, MARK4 and PKD) (4).

References

1. Abderrazak, A., Syrovets, T., Couchie, D., El Hadri, K., Friguet, B., Simmet, T., & Rouis, M. (2015). NLRP3 inflammasome: From a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biology. https://doi.org/10.1016/j.redox.2015.01.008

2. Yang, Y., Wang, H., Kouadir, M., Song, H., & Shi, F. (2019). Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death and Disease. https://doi.org/10.1038/s41419-019-1413-8

3. Zahid, A., Li, B., Kombe, A. J. K., Jin, T., & Tao, J. (2019). Pharmacological inhibitors of the nlrp3 inflammasome. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2019.02538

4. Kelley, N., Jeltema, D., Duan, Y., & He, Y. (2019). The NLRP3 inflammasome: An overview of mechanisms of activation and regulation. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms20133328

Long Name

NLR Family, Pyrin Domain Containing 2

Alternate Names

AGTAVPRL, AII, CIAS1, Cryopyrin, FCAS, FCU, MWS, NACHT, NALP3, PYPAF1

Gene Symbol

NLRP3

Additional NLRP3/NALP3 Products

Product Documents for Recombinant Human NLRP3/NALP3 GST (N-Term) Protein

Product Datasheets

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COA

SDS

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Product Specific Notices for Recombinant Human NLRP3/NALP3 GST (N-Term) Protein

This product is produced by and distributed for Abnova, a company based in Taiwan.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. This product is guaranteed for 1 year from date of receipt.

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