Technique / Immunology / Antibody

epitope mapping (4)

Preparation Of Peptide-KLH Conjugates For Immunization recommended protocol
A very successful protocol written by Claire Walczak in Mitchinson lab at Harvard medical school. ...

coupling antibodies to protein A/G protocol
coupling antibodies to protein A/G protocol from Breeden's lab protocols. ...

Polyclonal Antibody Production protocol
Very useful for rapid and simple generation of antibodies for western blots, ELISA assays, and immunoprecipitation. From Kitto lab at The University of Texas. ...

Making antibodies to synthetic peptides protocol
You'll need to couple the synthetic peptide hapten to a protein carrier, with a bifunctional reagent. Protocol from Daniel T. O'Connor, UCSD. ...

Monoclonal Antibody Production protocol
Illustration of monoclonal antibody production flowchart. A detailed protocol on Monoclonal Antibody Production Protocol maintained by Natural Toxins Research Center at Texas A&M University - Kingsville ...

Protocols for the conjugation of monclonal antibody to fluorochromes protocol
In this series of web pages, protocols, notes, and various illustrations are given to aid in the conjugation of proteins--principally monoclonal antibodies--to fluorescent dyes. (Mario Roederer) ...

Testing Rabbit Bleeds By ELISA protocol
By Claire Walczak 1996 version. You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by doing ELISAs against peptide conjugated to BSA. By conjugating to BSA, you will eliminate any signal for ...

Coupling Peptides To Resin For Affinity Purification protocol
Written by Claire Walczak (1996) in Michison lab at Harvard medical school. ...

Purification Of Anti Peptide Antibody protocol
Claire Walczak, Mitchinson lab at Harvard medical school (1996 version). ...

Hybridoma production protocol new protocol
Hybridoma monoclonal antibody production protocol. (Dr. Bart Frank, Arthritis and Immunology Program, OMRF) Reference: Ducey, C. M. and Frank, M. B. Hybridoma Production. In: Frank, M. B. ed. Molecular Biology Protocols. ( ...

Affinity Purified Monospecific Antibodies to Gel-Purified Proteins protocol
Since the introduction of sensitive immune assays, it has become increasingly apparent that previous criteria of protein purity are inadequate for the production of truly specific antisera. This method investigate the feasibility of using preparative electrobl ...

Antibody cleanup protocol
In order to decrease the amount of nonspecific staining, it is often necessary to preabsorb primary and secondary antibodies to yeast cells lacking the antigen prior to use. A 1:1 mixture of fixed yeast whole cells and spheroplasts are used for this purpose. ( ...

Purification of  Monoclonal Antibody from Hybridoma Culture Supernatant new protocol
Two steps of Purification of  Monoclonal Antibody from Hybridoma Culture Supernatant. (Wolf Lab, Harvard School of Public Health) 1. Hyridoma cell culture 2. Affinity purification of antibody. ...

Fusion protocol for monoclonal antibody production protocol
Protocol from Lab research in immunology at University of Louvain, Belgium. Fusion is an important procedure to produce monoclonal antibodies. (with scheme table) ...

GST-fusion protein injection into rabbits protocol
Protocol of injection of GST-fusion protein into rabbits for raising polyclonal antibodies. (Dr. Ivan Orlic) ...

Biotinylation of Antibodies protocol
This protocol is based on an original protocol devised by Aaron Kantor. Biotin is used in two-step detection systems in concert with conjugated avidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 3 and 6 biot ...

Conjugation of monoclonal antibodies protocol
Protocol from M. Roederer. In this series of web pages, protocols, notes, and various illustrations are given to aid in the conjugation of proteins--principally monoclonal antibodies--to fluorescent dyes. These conjugation procedures are commonly performed in ...

Antibody Labeling protocol
Thin sections of biological material, mounted on specimen grids, can be easily labeled by floating them, section-side down, on small drops of antibody. This method will work for sections through resin-embedded material as well as for thin, thawed, cryosections ...

Preparing Serum from Whole Blood hot protocol
A short protocol on serum preparation from whole blood by Michael Koelle, from Harlow and Lane "Antibodies: A Laboratory Manual". (Koelle lab, Yale University) ...

Affinity Purification of Antibodies Using Proteins Bound to Nitrocellulose new protocol
Protocol for affinity purification of antibodies using proteins bound to nitrocellulose by Michael Koelle, adapted from Will Talbot & Frank Solomon's lab. ...

Antibody purification using protein A/G column or peptide affinity column new protocol
A detailed protocol on antibody purification using protein G (monoclonal) or A (polyclonal) column or peptide affinity column. (Morris White's lab, Howard Hughes Medical Institute, Division of Endocrinology, Children's Hospital Boston, Harvard Medical School) ...

Antibody structure new review
A mini review on antibody structure including a closer look at antibody/antigen Interaction. It's the basis for many troubleshootings. ...

The Study of Antibody Recognition new review
A review on antibody recognition study (from antibody resource page). ...

Tech notes on Antigen Design & Sera Purification review
Antigen Design & Sera Purification Tech Sheet from Sigma Genosys. This tech sheet will briefly explore peptide selection and design, coupling strategy, and carrier proteins which are important factors in anti-peptide antisera generation. Serum purification ...

Overview of Antibody Production and tips review
Overview of Antibody Production and tips by AnaSpec, Inc. Content: Introduction Antigen Selection Synthetic Peptides as Antigens Selection of Animals Sampling Serum Complement Inactivation Storage of Antibodies References Tips for ...

Antibodies protocol guide new review
A tech book from Clontech on antibodies application. (Clontech) Table of Contents: I. Introduction 3 II. Materials Required 3 III. Western Blotting 4 A. Preparation of Cell Lysate and Electrophoresis 4 B. Western Blotting 5 IV. Immunoprecipit ...

Some useful info on how to select an antigenic peptide for antibody production site
This page can be used as a general flow chart on how to select a antigenic peptide to be synthesized for polyclonal antibody production. (ADI) ...

Ascites in Rabbits? troubleshooting
I have developed a rabbit-rabbit (rabbit plasmacytoma cell line fused with rabbit splenocytes) monoclonal antibody and wanted to know if you can produce ascites in rabbits practically? I know ascites production is routintely done in mice, but has an ...

Peptide Antisera Frequently Asked Questions troubleshooting
Peptide Antisera Frequently Asked Questions from Sigma Genosys. Content: General Questions Animal Welfare Conjugation Immunization Sodium Azide Antibody Concentration Antisera Purification Serum Storage & Handling Western Blot FAQ ...

Peptide antisera troubleshooting new troubleshooting
A troubleshooting guide on antisera production using peptide antigen. (From Sigma-Genosys Canada). Content: My serum looks cloudy, is it okay? I have received my sera and they are red in color. What is this and do I need to be concerned? I am ...

The world's antibody gateway new database
The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antib ...

Antibody search engine database
Antibody search engine Antibody search engine for all commercially available antibodies from over 300 antibody suppliers. Antibody search and bro ...

Production of monoclonal antibodies.
(Curr Protoc Immunol. 2006 Sep;Chapter 2:Unit 2.5.)
Yokoyama WM, Christensen M, Santos GD, Miller D.

Washington University School of Medicine, St. Louis, Missouri, USA.

This unit describes the production of monoclonal antibodies beginning with
immunization and cell fusion and selection. Support protocols are provided for
screening primary hybridoma supernatants for antibodies of desired specificity,
establishment of stable hybridoma lines, cloning of these B cell lines by
limiting dilution to obtain monoclonal lines, and preparation of
cloning/expansion medium. An alternate protocol describes cell fusion and
one-step selection and cloning of hybridomas utilizing a semi-solid
methylcellulose-based medium (ClonaCell-HY).

Antibody protocols from Nature Protocols.
(Nat Protoc.)
Nat Protoc. 2007;2(10):2574-81.

Production of a site- and phosphorylation state-specific antibody.

Goto H, Inagaki M.

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi
464-8681, Japan.

Protein phosphorylation plays important roles in various aspects of cellular
events. Visualization of site-specific phosphorylation in cells is of great
importance not only to analyze spatial and temporal distribution but also to
investigate biological function. Now, site- and phosphorylation state-specific
antibodies are widely utilized as the most powerful tools for these analyses.
This protocol details a method to produce the polyclonal version of such an
antibody by immunizing a synthetic phosphopeptide corresponding to a protein
phosphorylated at targeted site(s). This protocol is also applicable to the
production of other types of antibodies, which specifically recognize the
site-specific modification, such as acetylation, methylation and proteolysis. The
protocol can be completed in 2-3 months.

2: Nat Protoc. 2007;2(7):1763-9.

Purification of antibodies using the synthetic affinity ligand absorbent
MAbsorbent A2P.

Chhatre S, Titchener-Hooker NJ, Newcombe AR, Keshavarz-Moore E.

Department of Biochemical Engineering, University College London, Torrington
Place, London WC1E 7JE, UK.

A protocol for the purification of polyclonal antibodies from ovine serum using
the synthetic protein A absorbent MAbsorbent A2P is described. Clarified serum is
loaded directly onto the affinity column without prior adjustment and albumin and
unwanted serum components are washed from the column using a sodium octanoate
buffer before elution of bound antibodies. MAbsorbent A2P was shown to bind
approximately 27 mg ml(-1) of polyclonal immunoglobulin under overloading
conditions, with eluted IgG purities of >90% and minor levels of albumin
(approximately 1%). The anticipated time required to complete the purification
protocol is 6-7 h. Although the protocol is similar to methods utilized for
antibody purification using chromatography with protein A derived from the cell
wall of the microorganism Staphylococcus aureus or protein G from Streptococcus
as the affinity ligands, affinity absorbents based on synthetic ligands offer a
number of advantages to compounds derived from biological sources, in particular
robustness, relatively low cost, ease of sanitization and, in principle, lack of
biological contamination.

Antibody technique books
(Biowww bookshelf)
Antibody method and protocol books

Figure. Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen
(Molecular Biology of the Cell)
III. Methods 8. Manipulating Proteins, DNA, and RNA Isolating Cells and Growing Them in Culture

Antibody-antigen interactions
(Molecular Biology of the Cell)
V. Cells in Their Social Context 24. The Adaptive Immune System B Cells and Antibodies

Figure. Procedure for producing a monoclonal antibody
(Molecular Cell Biology)
6. Manipulating Cells and Viruses in Culture 6.2. Growth of Animal Cells in Culture

Storage of Antibodies
best way of store antibodies for Westerns

Hybridoma stability
fusions with sp2/0 to make a hybridoma

antibody generation from rat
not got any response after the second boost in rats

Method to freeze hybridoma
hybridomas from transgenic mouse spleen

Raising Antibody problems
problem in raising antibody against 35Kd recombinant protein in Rabbit

Hybridoma production troubleshooting
mouse spleen and sp2 cell fusion problem

preabsorbing chicken antibodies
chicken primary antibody that preabsorbed against it's antigenic peptide


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