We would like to be able to screen multiple signaling pathways at once with primary cells after stimulating with whatever stimulus we choose. Doing tons of western blots is not possible so hoping someone can provide an answer to what they have used to look at for example JAK/STAT, MAPK, PI3K, NFkB, etc all at once. So far I've seen R&D has a phosho-kinase array but do need some feedback for what has worked well for primary cells. Phosphorylation flow cytometry is also a possibility but would rather do a broad screen first to give us a clue of what pathways to study further. Thanks!
We pool primary cells and run cytokine arrays on them- you can mix and match the signals you'd like to look for. They're very expensive but you very quickly see activation patterns in the different groups. They can also be finiky- so practice makes perfect. (note that the link is for a kit I haven't used, not the same as the publication).
Looks like NioRad has a full line of pathway PCR kits- it may be worthwhile to to talk to them.
https://www.fullmoonbio.com/product/jak-stat-phospho-antibody-array/
http://www.bio-rad.com/en-us/prime-pcr-assays/pathway/primepcr-pathways#J100006-C38DA583-3787-48B9-B8B1-9A649843BDC8
Article Type I IFNs Act upon Hematopoietic Progenitors To Protect an...
Thanks for your quick reply. My only concern was how many primary cells are needed to do any of these signaling arrays to get enough lysate? I will see in your paper if those details are there and also contact the company. Thanks again
We worked with anemic mice and required about 6 mice per group. Luckily the arrays are very sensitive!
Hi Deanna,
Because a lot of signalling pathways elements are constitutively expressed and activated at a post-translational levels (Phosphorylation, Acetylation etc..) it can also be useful to analyse your protein samples using proteome Profiler array. These are membrane with cross-linked antibodies directed against various RTK phosphorylation as well as MAP Kinase and NF-KB. I used in the past kit from R&D biotechnology and they work pretty well.
https://www.rndsystems.com/products/proteome-profiler-antibody-arrays
Check out other company as well because they may have other pathways you are more interested in.
All the best,
Karl
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