I have tried to detect ASC oligomers after overexpression but still struggling.
I can see ASC monomers and just smears somewhere above 100 kDa, but not dimers, trimers and oligomers in WB.
Any suggestions for troubleshooting?
Here are my procedures.
293T cells (10^6 per well on a 6-well plate) were transfected with 1 microgram of each plasmid of NLRP3 and ASC (expression of tagged proteins were confirmed).
For ASC oligomerization by crosslinking,
1. Wash the cells and lyse the cells with 100 microliter of Buffer A.
2. Shear the lysates with 20 times through 27 G needle.
3. Centrifuge at 340 g for 8 min at 4 °C.
4. Collect supernatant and add 1 vol. CHAPS buffer
5. Centrifuge at 2650 g for 8 min at 4 °C.
6. Resuspend the pellet in 20 microliter of CHAPS buffer containing 10 mM BS3 at room temperature for 30 min.
7. Add 5X SDS sample buffer and boil.
Then, Western blot with a 10% gel.
The assay was done according to this reference (except for the use of different crosslinking reagent).
https://www.researchgate.net/publication/271223869_Syk_is_involved_in_NLRP3_inflammasome-mediated_caspase-1_activation_through_adaptor_ASC_phosphorylation_and_enhanced_oligomerization
Buffer A and CHAPS buffer were prepared according to this reference.
https://www.researchgate.net/publication/251452825_Chapter_Thirteen_Assembly_Purification_and_Assay_of_the_Activity_of_the_ASC_Pyroptosome
I've also done based on this paper, but in vain.
https://www.researchgate.net/publication/264988115_Influenza_A_virus_protein_PB1-F2_translocates_into_mitochondria_via_Tom40_channels_and_impairs_innate_immunity
Article Syk is involved in NLRP3 inflammasome-mediated caspase-1 act...
Article Chapter Thirteen Assembly, Purification, and Assay of the Ac...
Article Influenza A virus protein PB1-F2 translocates into mitochond...
Dear Hong Su,
Hi. I am also facing the same problem. Have got smear sometimes but not a clear band yet. Please let me know if you come up with any solution. If anyone else can suggest some thing to troubleshoot the problem, it would be a great help. Thank you.
BS3 is not an ASC-specific crosslinking reagent and it can cross link, without preference, any two proteins with primary amines. This also means that your ASC may be getting cross-linked with other non-ASC proteins, which makes that cross-linked complex detectable via anti-ASC antibodies (since ASC is apart of that complex) while not exactly depicting dimers or trimers of ASC.
Hi, Jatin.
BS3 has been successfully used for the ASC oligomerization in many articles or protocols, which led me to try with it.
I also tested with DSS (used in other articles) but did not work.
I believe there must be a critical step for this assay to work (that are not briefly revealed in the materials/methods of those articles) and I have been looking for what that is.
If BS3 is not an ASC-specific agent, how could it allow the people to produce good results in those papers, in your thought? What can be suggested as key points to success (either with BS3 or other agents you may be able to recommend)?
Hong-su Park you add one volume of CHAPS buffer, can you see pellet after you centrifuge it?
Hi Hong-su Park ,when I add DSS(disolved in DMSO first) into the PBS, it separated out from PBS immediatly. After crosslinking , DSS was spin down with ASC. The problem is DSS is insoluble, it has to be loaded in the SDS-PAGE with crosslinked protein. Then, the insloluble DSS jam the gel, disturbe the seperation of proteins.How can I solve this problem?
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