Splenic nuclear protein extract was prepared using the Nuclear Extract Kit (#40010) from ActiveMotif (Carlsbad, CA). Because the most common form of NFκB is the heterodimer consisting of p65/p50, p65’s DNA-binding activity was assessed in the spleen as a measure of activated NFκB using the TransAM NFκB p65 Chemi kit (#40097, ActiveMotif, Carlsbad, CA). Recombinant p65 protein (#31102) was purchased from ActiveMotif (Carlsbad, CA) and used to create a standard curve.
Transam nfκb p65 chemi kit
Manufactured by Active Motif
The TransAM NFκB p65 Chemi kit is a chemiluminescent-based assay designed to detect and quantify the activated form of the NF-κB p65 subunit in nuclear extracts. The kit provides a simple, sensitive, and high-throughput method for measuring NF-κB p65 activation.
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2 protocols using transam nfκb p65 chemi kit
1
Splenic NFκB p65 DNA-Binding Assay
2
NF-κB p65 Activation Assay in CLL
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The TransAM® NF-κB p65 Chemi kit (Active Motif, Carlsbad, CA) was used to detect NF-κB activity. TG02 or Lcki were added 1 h prior to anti-IgM, and the CLL cells were collected 2 h later. Two microgram nuclear extracts were used for p65 activation analysis by ELISA per kit instruction. Nuclear extract from TPA and calcium ionophore activated Jurkat cells were used as positive control. The wild-type and mutated p65 consensus binding oligonucleotides were used to monitor the specificity of the analysis. The chemiluminescence was read with a Synergy2 SL Luminescence Microplate Reader (BioTek, Winooski, VT).
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