Development, Evaluation, and Application of a Highly Sensitive Microtiter Plate ELISA for Human Bcl10 Protein

Abstract

Bcl10 (B-cell CLL/lymphoma 10) is a 233 amino acid CARD (caspase recruitment domain)-containing cellular protein, increasingly recognized as a mediator of NFkappaB activation in non-immune, as well as immune cells. Due to the importance of Bcl10 in diverse cell types, we developed a solid-phase, enzyme-linked immunosorbent (ELISA) assay to precisely measure Bcl10 in small volume cell lysates, using recombinant Bcl10 to standardize the assay. Standard curve measures Bcl10 from 0.25 ng/mL to 16 ng/mL, with very low intra- and inter-assay variation. Sample dilution and exogenous Bcl10 recovery experiments, comparisons with Western blot, and linear response to increasing doses of known Bcl10 activators confirm the specificity and precision of the ELISA.

Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1

“…The effectiveness of silencing in the HepG2 cells was demonstrated by Western blotting for TLR4 and by quantitative ELISAs for BCL10 (1,33) and for GRB10 (Blue Gene, Shanghai, China). Briefly, cells were grown to 60 -70% confluency in 6-well tissue culture clusters, and the medium of the growing cells was aspirated and replaced with 2.3 ml of fresh medium with serum.…”

Tumor Necrosis Factor α-induced Inflammation Is Increased but Apoptosis Is Inhibited by Common Food Additive Carrageenan

“…After 24 h, the spent medium was exchanged with fresh growth medium. The effectiveness of silencing of BCL10 expression was determined by ELISA assay and Western blot (12,37).…”

Specific effects of BCL10 Serine mutations on phosphorylations in canonical and noncanonical pathways of NF-κB activation following carrageenan

“…Expression of BCL10 protein in control and CGN-treated NCM460 was determined by a solid-phase ELISA, developed for quantitative determination of BCL10 (8). Control or treated cells were lysed in RIPA buffer (50 mM Tris containing 0.15 M NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS, pH 7.4), and the cell extracts were stored at Ϫ80°C until assayed by the ELISA.…”