Agarose beads

An important factor in all these experiments is the choice of bead used to immobilize the probe. Biochemists have considered cross-linked agarose beads to be exceptionally hydrophilic with a low tendency to bind proteins nonspecifically, and these beads have the further attraction of being commercially available in activated forms (succinimidyl esters, epoxides, and maleimides, for example). However, early trials of bead-based chemical proteomics have shown that many proteins in mammalian cell lysates bind tenaciously to agarose beads. This was unimportant in many studies in which protein-protein interactions were detected by coimmunoprecipitation with immunochemical... 

Wash 3 times for 5 min with buffer and collect agarose beads by centrifuging at 2000 re/followed by aspiration of buffer... 

Streefkerk JG, van der Ploeg M, van Duijn P. Agarose beads as matrices for proteins in cytophotometric investigations of immunohistoperoxidase procedures. /. Histochem. Cytochem. 1975 23 243-250. 

In another application of coupling proteins to surfaces using click chemistry, Duckworth et al. (2006) carried out prenylation of a protein using a farnesyl azide derivative and the enzyme farnesyl transferase for subsequent chemoselective ligation to alkyne-functionalized agarose beads. The result is a highly discrete, site-specific attachment of the protein to the solid phase at a single location. 

Inman, J.K. (1985) Functionalization of agarose beads via carboxymethyladon and aminoethylamide formation. In Affinity Chromatography-A Practical Approach (P.D.G. Dean, W.S. Johnson, and F.A. Middle, eds.), pp. 53-59. IRL Press, Washington, DC. 

Figure 6.14 Schematic representation of the principle of biospecific affinity chromatography. The chosen affinity ligand is chemically attached to the support matrix (agarose bead) via a suitable spacer arm. Only those ligands in solution that exhibit biospecific affinity for the immobilized species will be retained...

Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. 

While the binding of aminoglycosides to the RRE provides a proof of principle, their affinity and, in particular, selectivity traits need to be improved for true therapeutic utility. To facilitate the discovery of potent and selective RRE binders, we developed a solid-phase assay. The components of this assembly include (a) insoluble agarose beads (or microtiter plates) covalently modified with streptavidin, (b) a biotinylated RRE fragment, and (c) a fluorescein-labeled Rev fragment (RevFl). Assembly of the three components generates an immobilized ternary complex whereby the biotinylated RRE binds to the beaded... 

It should be mentioned here that another family of hydrocarbonaceous phases has been developed for the hydrophobic chromatography of biopolymers 49, 50). For the preparation of such stationary phases agarose beads are first activated with cyanogen bromide and subsequently reacted... 

Derivatized silica gel that is used for size-exclusion HPLC contains a proprietary bonded phase that is defined as hydrophilic to minimize nonspecific hydrophobic and ionic interactions. Polymeric supports consist of highly cross-linked agarose beads, with or without bonded dextran or cross-linked copolymers of allyl dextran and AyV -methylenebisacryl-amide. All supports are available with a variety of particle and pore sizes and distribution. 

Both IgG and Csb were found to stimulate the respiratory burst separately and independently in one study but in another was active only in the presence of IgG. The divergent findings may be attributable to differing methods. In one study serum was mixed with agarose beads which fix complement with the binding of Csb- One set of beads was then heated at 50° for 30 min to remove 35 and the other was boiled in 2 M NaCl to remove IgG. The completeness of the removal of each component was verified by the loss of reactivity of the beads with a fluorescent monospecific antibody. Beads prepared in this way with either IgG or with Cjb attached elicited the formation of O by PMNs whereas the release of lysosomal contents occurred only with beads containing both IgG and Cab. The effects of the IgG-agarose were blocked by Ffab lj and those of Cjb were blocked by antiserum to C,. 

Clearly a number of differences could explain these divergent results. Boiling could cause loss of antigenicity but retention of activity in stimulating the formation of O . Alternately, the Cab might be attached to agarose beads and to erythrocytes in different ways so that different parts of the molecule were exposed to the PMNs. The assays used to detect contamination of Cab by IgG may also differ in sensitivity (immunofluorescence vs. gel electrophoresis of isolated Ca). 

An example of a successful application of affinity chromatography is the isolation of the enzyme cytidine deaminase from cells of E. coli. Cytidine was linked covalently via long spacer arms to the agarose beads as in the following diagram ... 

Four types of media possess most of these desirable characteristics agarose, polyvinyl, polyacrylamide, and controlled-porosity glass (CPG) beads. Highly porous agarose beads such as Sepharose 4B (Pharmacia) and Bio-Gel A-150 m (Bio-Rad Laboratories) have virtually all of these characteristics and are the most widely used matrices. Polyacrylamide gels such as Bio-Gel P-300 (Bio-Rad) display many of the recommended features however, the porosity is not especially high. 

Ninhydrin (1,2,3-triketohydrindene monohydrate). Widely available chemical, light-sensitive. Ninhydrin reacts with free amines (2 1 molar ratio), giving a purple product (Ruhemann s purple resonance structure). Used for a qualitative test to determine the presence of aliphatic amines on the agarose beads as a 0.2% w/v solution in ethanol (see Note 4). Hazards Harmful if swallowed skin, eye, and respiratory irritant. Toxicity data LD50 78 mg/kg intraperitoneal, mouse. Should be handled in a fume hood with safety glasses and gloves. 

Amination of agarose beads. The washed epoxy-activated gel is suspended in 1 mL of distilled water/g of gel in a 1-L conical flask. About 1.5 mL of ammonia/ g of gel are added and the gel incubated for 12 h at 30°C in a rotary shaker. Alternatively, 1,6-diaminohexanediamine (5 eq.) can be used to aminate the gel, yielding resins with a six-carbon spacer arm. The aminated gel is washed with 40 mL of distilled water/g of gel on a sinter funnel, and stored in 20% v/v ethanol at 0-4°C. The extent of amination is determined as described in Note 3. Aminated beads can also be purchased from Amersham Biosciences. 

Extent of epoxyactivation of agarose beads. Sodium thiosulphate (1.3 M, 3 mL) is added to 1 g of epoxy-activated gel and incubated at room temperature for 20 min. This mixture is neutralized with 0.1 MHC1 and the amount of HO used is recorded. The volume of 0.1 MHO added corresponds to the number of OH moles released (10 pmol for each 100 pL added), which equals the pmol of epoxy groups/g gel. Therefore, the extent of epoxy activation is expressed as pL HC1 used/10 pmol/g gel. The protocol usually results in 25 pmol epoxy groups/g moist weight gel. 

Fig. 17 Pattern of DNP-Ab-functionalized agarose beads on a glass support functionalized with a dinitrospiropyran photoisomerizable layer.

Scheme 18 Photochemical patterning of a photoisomerizable dinitrospiropyran monolayer associated with a glass support with agarose beads functionalized with DNP-Ab.

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