Abstract
B lymphocyte activation is regulated by its membrane-bound B cell receptors (BCRs) upon recognizing diverse antigens. It is hypothesized that antigen binding would trigger conformational changes within BCRs, followed by a series of downstream signaling activation. To measure the BCR conformational changes in live cells, a fluorescent site-specific labeling technique is preferred. Genetically encoded fluorescent tags visualize the location of the target proteins. However, these fluorescent proteins are large (~30 kDa) and would potentially perturb the conformation of BCRs. Here, we describe the general procedures of utilizing short tag-based site-specific labeling methodologies combining with fluorescence resonance energy transfer (FRET) assay to monitor the conformational changes within BCR extracellular domains upon antigen engagement.
Highlights
[Background] B lymphocytes are responsible for the production of protective antibodies against pathology arising from the recognition of antigens by the cell membrane expressed B cell receptors (BCRs)
fluorescein arsenical hairpin binder (FlAsH) emits green-yellow fluorescence, while the color variant, resorufin arsenical hairpin binder (ReAsH) fluorescence peaks in the red (609 nm), would serve as a Fluorescence resonance energy transfer (FRET) acceptor for green fluorescent signal, such as Coenzyme A (CoA)-488
We describe the procedures to label the BCRs and soluble Ig by inserting the ybbR tag and tetracysteine tag in BCR molecule, as well as the usage of necessary components to set up the site-special labeling system
Summary
[Background] B lymphocytes are responsible for the production of protective antibodies against pathology arising from the recognition of antigens by the cell membrane expressed B cell receptors (BCRs). In terms of the heavy chain of IgM-BCR, it includes 4 domains, Cμ1, Cμ2, Cμ3, and Cμ4 (Figure 1). Variable domains in the heavy and light chain polypeptides form an antigen-binding site unique to the antibody.
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