Labeling Antibodies.

This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies.

Purification of Labeled Antibodies Using Size-Exclusion Chromatography.

Many antibody labeling procedures call for a desalting or purification step requiring size-exclusion chromatography (SEC). The method outlined here contains information needed to desalt an antibody conjugate. Similar procedures would be used for ion-exchange chromatography using a gradient of increasing ionic strength.

Iodination of Antibodies with Immobilized Iodogen.

Iodination, a chemical or enzymatic incorporation of I to specific amino acid side chains, is a commonly used method for labeling antibodies with radioisotopes. Commercially available products make iodination of antibodies a simple and quick process. One example, used here and available at Pierce, is the "Iodination bead," or -chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads.

Labeling Antibodies Using Colloidal Gold.

Colloidal gold-antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl, is reduced with dilute solutions of sodium citrate, as described here.

Labeling Antibodies Using Europium.

There are many uses for antibodies labeled with metal ions. Most of these methods involve first attaching a metal chelator to the antibody molecule. This is achieved using standard cross-linking chemistry and then adding the desired metal at appropriate concentration and pH.

Biotinylating Antibodies Using Biotin-Long Chain (LC) Hydrazide.

Hydrazide derivatives are useful for biotinylating antibodies at oxidized carbohydrate groups. This protocol uses oxidation conditions that will convert most if not all possible hydroxyl sites to aldehydes. Each antibody may require different oxidation conditions to optimize labeling.

Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide.

There are several techniques for biotinylating antibodies, from the most basic (using NHS-ester biotin to label primary amines) to more complex experiments (modifying sulfhydryls and carbohydrates). Biotinylation of free sulfhydryls, described here, can be effectively mediated using haloacetyl biotin derivatives. To modify an antibody using this reagent, sulfhydryls must be available.

Labeling Antibodies with -Hydroxysuccinimide-Long Chain (NHS-LC)-Biotin.

Labeling antibodies with biotin (biotinylation) is a useful and simple technique. Biotin's small size (244 Da) usually has little effect on the biological activity of the protein target. The most common way to biotinylate an antibody is to cross-link a biotin succinimidyl ester to a primary amine.

Labeling Antibodies with Cy5-Phycoerythrin.

Conjugates of the FRET dye Cy5-phycoerythrin (Cy5PE) with antibodies are relatively straightforward to make. The protocol does require synthesis of the Cy5PE tandem dye. Phycoerythrin (PE) can be purchased from multiple vendors.

Conjugation of Antibodies to Horseradish Peroxidase.

Antibodies conjugated with horseradish peroxidase (HRP) are one of the most widely used bioreagents in the biological sciences. This protocol is a basic method for adding HRP to a thiolated antibody and can be adapted for use with different cross-linkers. Conjugation methods usually focus on linking through the lysines on HRP because there are only six of them and their modification does not adversely affect enzyme activity.

Antibody Purification and Storage.

Antibodies have become a common and necessary tool in biochemistry, cell biology, and immunology laboratories. There are many different types of antibodies and antibody fragments being used for a myriad of applications. As a result, many different purification protocols have been developed to obtain antibodies of the desired specificity and sensitivity.

Peptide Affinity Purification of Antibodies.

This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers.

Conjugation of Peptides to Thiol-Reactive Gel for Affinity Purification of Antibodies.

Thiol-reactive linkers, such as iodoacetyl or maleimide, bound to cross-linked agarose are used to attach cysteine-containing peptides covalently to this resin for use in affinity-purification protocols. It is critical to confirm that the peptide contains a reduced cysteine so that the thiol is available for conjugation to the thiol-reactive linker. The column should be sized appropriately for the amount of peptide to be used and the volume of serum to be processed.

Labeling Antibodies Using a Maleimido Dye.

Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.5-7.

Labeling Antibodies Using -Hydroxysuccinimide (NHS)-Fluorescein.

-Hydroxysuccinimide (NHS)-ester derivatives are among the most commonly used reagents for labeling proteins. The method described here can be adapted to use practically any NHS fluorophore. Generally, a fluorophore is covalently bound to a macromolecule such as an antibody and acts as a reporter molecule used to measure the presence of the macromolecule.

Protein A and Protein G Purification of Antibodies.

Protein A and Protein G are immunoglobulin-binding proteins expressed in and sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation.

Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography.

Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method.

Isolation of IgY from Chicken Eggs.

Because IgY does not interact with either Protein A or Protein G, more traditional methods must be used for its isolation. To isolate chicken IgY from the yolk of chicken eggs, the yolks and the whites must be separated much in the way a cook does. Minimizing the egg white proteins is an important part of this process.

Preparation of Antibody Using Caprylic Acid.

Caprylic acid has been used to enrich IgG from serum, ascites, and cell culture supernatants by precipitating the non-IgG serum proteins. By precipitating all of the unwanted serum proteins rather than the antibodies, the tendency of antibodies to aggregate when precipitated is avoided. This method should not be used with antibody sources that contain low concentrations of antibody, such as many cell culture supernatants, owing to the potential loss of high-affinity antibodies, which may be bound by the caprylic acid.

Ammonium Sulfate Fractionation of Antibodies.

Before the widespread availability and use of Protein A (rabbit) or Protein G (rodent) for the purification of IgG, the use of an ammonium sulfate "cut" was the standard method to isolate IgG and other serum proteins. The addition of ammonium sulfate reduces the effective solubility of proteins through direct competition for binding sites on the surface of the protein. The resulting precipitated proteins can be isolated by centrifugation.

VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available.

Pyroglutamate-Aβ 3 and 11 colocalize in amyloid plaques in Alzheimer's disease cerebral cortex with pyroglutamate-Aβ 11 forming the central core.

N-terminal truncated amyloid beta (Aβ) derivatives, especially the forms having pyroglutamate at the 3 position (AβpE3) or at the 11 position (AβpE11) have become the topic of considerable study. AβpE3 is known to make up a substantial portion of the Aβ species in senile plaques while AβpE11 has received less attention. We have generated very specific polyclonal antibodies against both species.

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells.

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics.

Medical care needs of returning veterans with PTSD: their other burden.

Background: There has been considerable focus on the burden of mental illness (including post-traumatic stress disorder, PTSD) in returning Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF) veterans, but little attention to the burden of medical illness in those with PTSD.

Objectives: (1) Determine whether the burden of medical illness is higher in women and men OEF/OIF veterans with PTSD than in those with No Mental Health Conditions (MHC). (2) Identify conditions common in those with PTSD.

Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2delta6.

Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized.