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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.
NLRP3 can be challenging target to work with in western blot.
NLRP3 is regulated by post-translational modifications, such as ubiquitination and phosphorylation. It has several isoforms in human and mouse. Therefore, it’s may exist several bands in a western blot assay. Moreover, the predicted molecular weight of NLRP3 is about 118 kDa. The 8% gel is more appropriate. Large proteins (>100 kDa) will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will facilitate transfer. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also prevent precipitation. The observed band size of NLRP3 may not be the same as predicted MWs in western blot due to the different forms of the target.
|Sample preparation||Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation|
|Ultrasonicate samples to enrich more target proteins|
|Keep samples on ice during the whole WB process|
|Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.|
|Electrophoresis||For large proteins (the MW of target protein >100 kDa), be sure to run samples in 8% or lower separating gel.|
|Transferring||It is preferred to add SDS to a final concentration of 0.1% in the transfer buffer for large proteins.|
|Wash PVDF membrane to remove methanol completely.|
|To determine if the transfer is successful by visualization of proteins in membranes using Ponceau S.|
|Maximizing signal||The level of NLRP3 expression can be elevated when cells are treated with LPS1|
|There may be multiple bands of NLRP3 in WB assay. NLRP3 can be regulated at post-translational level, such as phosphorylation, ubiquitination. And NLRP3 has 6 isoforms in human(predicted).|
Human THP1 WT cells treated with 200 ng/ml LPS for 3h
Rat spleen lysate
Jurkat (human t cell leukaemia t lymphocyte) whole cell lysate
Find full information on working with NLRP3: