We plan to measure NFkB activation in fibroblasts in vitro without using western blots.
If anyone has an experience with the 2 following methods, which is preferable:
measuring phospho- vs total- p-65 or p-50 with ELISA kits
OR
measuring DNA binding of p-p65 or p-p50 with appropriate kits (e.g. TransAm)
Thank to all who respond.
If you do the ELISA for total vs. phosphorylated versions you will measure the amount of activated transcription factor. If you use a Transam kit (or similar), you will be measuring the amount of activated NFkB that actually is binding to its target sequence, so in both methods, you are measuring different things. Depends on what you want to know.
The TransAM assays for p65 are really popular (https://www.activemotif.com/catalog/216/transam-nf%CE%BAb-p50-p52-p65-family-kits), and there are over 100 publication using this kit (https://www.citeab.com/kits/3790855-40096-transam-nf-b-p65?utm_campaign=Widget+All+Citations&utm_medium=Widget&utm_source=Active+Motif&utm_term=Active+Motif).
There's also a promo for TransAM assays running right now, save 20% if you buy 2 or more of the same kit with promo code TRANSAM20. More details here: https://www.activemotif.com/promotions#TRANSAM20
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