Flow cytometry (FACS) Assay

Protocol-FACSFlow cytometry (FACS) Assay:. To date, a selection strategy
that imparts a growth advantage specifically to cells that express
beta-gal is not available. ...
www.stanford.edu/group/blau/protocols/facsprotocol.html

Protocol for cell sorter experiments

Dr. Aghoul
Department of Biological Sciences
Chicago State University

http://www.csu.edu/biologicalsciences/cytometry/flowcytomery.htm

Flow Cytometry Assay

Flow Cytometry Assay
to Measure Internalization of GPCR's

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

Expression mapping using a retroviral vector

Expression mapping using a retroviral vector for CD8+ T cell epitopes:
Definition of a Mycobacterium tuberculosis peptide presented by H2-Dd

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
&_user=2044985&_coverDate=03%2F31%2F2005&_rdoc=1&_fmt=&_orig=search&_sor
t=d&view=c&_acct=C000053655&_version=1&_urlVersion=0&_userid=2044985&md5
=8bc1f3bd34f16373f6dc997f1b7953e7

Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal

Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal ...

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

Epitope Mapping Using Synthetic Biotin-Labeled Peptides

Epitope Mapping Using Synthetic Biotin-Labeled Peptides
Ed Harlow and David Lane
CSH Protocols; 2006; doi:10.1101/pdb.prot4278 [Extract] [Full text]

Epitope Mapping of Hemagglutinating Adhesin

Epitope Mapping of

Epitope Mapping of. Hemagglutinating Adhesin. HA-Ag2 of ..... makes it unsuitable for the competition assay. MAb 31A10 ... iai.asm.org/cgi/reprint/60/7/2791.pdf

Epitope Mapping-Antibodies Protocols

Epitope Mapping-Antibodies Protocols - BiocompareEpitope Mapping By
Competition Assay-SUBSCRIPTION REQUIRED Source: CSH Protocols; 2006;
doi:10.1101/pdb.prot4277; Epitope Mapping Using Synthetic ...
www.biocompare.com/protocols/category/3772/Epitope-Mapping-Antibodies.ht
ml - 28k -

Epitope mapping for the anti-rabbit cholesteryl ester transfer ...

Epitope mapping for the anti-rabbit cholesteryl ester transfer ...

Relationship of the binding competition assay to the solid. phase binding assay for the ..... ever, the epitope mapping study showed no indication of ... www.jlr.org/cgi/reprint/40/11/2013.pdf -

Epitope Mapping by Mutagenesis

Epitope Mapping by Mutagenesis Distinguishes between the Two Tertiary
Structures of the Histidine-Containing Protein HPr
Sadhana Sharma, Fawzy Georges, Louis T. J. Delbaere, Jeremy S. Lee,
Rachel E. Klevit, E. Bruce Waygood
Proceedings of the National Academy of Sciences of the United States of
America, Vol. 88, No. 11 (Jun. 1, 1991), pp. 4877-4881 This article
consists of 5 page(s).
View Article Abstract

http://links.jstor.org/sici?sici=0027-8424(19910601)88%3A11%3C4877%3AEMB
MDB%3E2.0.CO%3B2-P

Epitope Mapping by Competition Assay

Epitope Mapping by Competition Assay

Ed Harlow and David Lane

ABSTRACT
The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can be used with antibodies that bind both conformational and linear epitopes, and it is most useful in the analysis of monoclonal antibody specificity because polyclonal sera typically recognize multiple different epitopes. This simple, ELISA-based protocol examines the ability of two hypothetical, newly derived monoclonal antibodies, XH1 and XH2, to compete with the well-characterized ZO-1 anti-YFP1 antibody for binding to the target antigen YFP1; hypothetical antibody PC10 is used as a control. First, 96-well plates are coated with the YFP1 antigen and direct binding assays are run to determine that all of the antibody preparations are active and able to react with the immobilized antigen. Second, the competition analysis itself is carried out. Note that the names of antibodies and antigens should be considered as generic.

Epitope Mapping by Competition Assay

Epitope Mapping by Competition Assay
Ed Harlow and David Lane
CSH Protocols; 2006; doi:10.1101/pdb.prot4277 [Extract] [Full text]

PCR-Based Analysis of Immunoprecipitated Chromatin

PCR-Based Analysis of Immunoprecipitated Chromatin
Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
CSH Protocols; 2007; doi:10.1101/pdb.prot4768 [Abstract] [Full text]

Chromatin Immunoprecipitation-ChIP

Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications
Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
CSH Protocols; 2007; doi:10.1101/pdb.prot4767 [Abstract] [Full text]

Immunofluorescence of Drosophila S2 Cells

An Approach for Immunofluorescence of Drosophila S2 Cells
Sarkar Angshuman and Schulz Cordula
CSH Protocols; 2007; doi:10.1101/pdb.prot4760 [Abstract] [Full text]

Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes

Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes
Rosa A. Uribe and Jeffrey M. Gross
CSH Protocols; 2007; doi:10.1101/pdb.prot4779 [Abstract] [Full text]

In Situ Hybridization and Immunohistochemistry

Baskets for In Situ Hybridization and Immunohistochemistry
Hazel L. Sive, Robert M. Grainger, and Richard M. Harland
CSH Protocols; 2007; doi:10.1101/pdb.prot4777 [Abstract] [Full text]

Preparation of Polyclonal and Monoclonal Antibodies

Polyclonal and Monoclonal Antibodies

Adaped from David Bowtell (bowtell@ariel.ucs.unimelb.edu.au)

http://mycoplasmas.vm.iastate.edu/lab_site/methods/immunol/poly_mono_abs
.html

Handbook of Immunochemical Staining Methods

Handbook of Immunochemical Staining Methods: Antibody (Dako)
Immunoglobulins / Polyclonal Antibodies / Monoclonal Antibodies /
Antibody Affinity / Antibody Cross-Reactivity / Antibody Reaction Rates
/ Antibody Stability / Handling of Antibodies / References
http://www.dakousa.com/ishbantibodies.pdf