Full Details
http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2007/406/pl5?etoc
http://www.csu.edu/biologicalsciences/cytometry/flowcytomery.htm
contributed by Patricia Tsao
Internalization of receptor is measured as the reduction of receptors on
the plasma membrane surface. Surface receptors are labeled with
fluoresent antibodies which is in turn measured using a flow cytometer.
The antibodies recognize an epitope engineered onto the N-terminus of
the GPCR.
The following protocol is optimized for internalization of FLAG-tagged
beta 2-adrenergic receptor (B2AR) stably transfected into HEK293 cells.
Full Protocol Here
http://www.ucsf.edu/vonzast/flow.html
http://209.85.135.104/custom?q=cache:-o87O5wI2JgJ:www.ucsf.edu/vonzast/f
low.html+Flow+Cytometry+protocol&hl=en&ct=clnk&cd=1&client=google-coop
Taiki Aoshi, Mina Suzuki, Masato Uchijima, Toshi Nagata and Yukio Koide,
Department of Microbiology and Immunology, Hamamatsu University School
of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan
Abstract
Identification of CD8+ T cell epitopes is important because detection of
specific CD8+ T cells after infection or immunization requires prior
knowledge of epitope specificity. Furthermore, identification of CD8+ T
cell epitopes permits the development of specific preventive and
therapeutic approaches to both infections and tumors. Thus far, CD8+ T
cell epitopes have been identified either using an overlapping peptide
library covering an entire protein, or using algorithms designed to
identify likely peptides that bind to major histocompatibility complex
(MHC) class I molecules. The synthesis of overlapping peptides can be
prohibitively expensive, and the algorithm programs used to predict CD8+
T cell epitopes are not always accurate. Here we describe a retroviral
expression system that specifically allows longer polypeptides and
shorter peptides to be expressed in the cytoplasm, and thereby to be
processed onto class I MHC molecules. T cells from mice that were
immunized with a DNA vaccine encoding MPT-51 were probed against
MHC-compatible cell lines retrovirally transduced with overlapping gene
fragments encoding 120-140 amino acids of the MPT-51 molecule. After
further testing of shorter peptide sequences, we identified a CD8+ T
cell epitope using cell lines expressing a relatively small number of
algorithm-predicted candidate epitopes. We found that one of the
requirements for cell surface display of the 20-mer peptide was the need
for cotranslational ubiquitination. The restriction molecule was
identified as Dd following transduction with MHC class I genes followed
by transduction with the oligonucleotide encoding the epitope. The
retroviral expression system described here is cost-effective,
particularly if the target molecule is large, and could be adapted to
identifying T cell epitopes recognized in infectious disease and against
tumor cell antigens.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-4FCSC6D-2
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| Epitope mapping of anti-D316-36 and anti-D320-33 antibodies. Biotin-labeled synthetic peptides (2 µg · ml - 1) were captured on streptavidin-coated ... iai.asm.org/cgi/content/full/68/3/1156 |
