Sepharose beads

Approximately 1 mg of total protein is pre-incubated with 25 /il of A-Sepharose beads CL-4B (Amersham Pharmacia Biotech) in 300 /d of binding buffer FLA for 1 h at 4° with gende rocking. The sample is then spun down at 1000 rpm for 3 min in an Eppendorf F 45-24-11 rotor, preserving the supernatant for further use. An aliquot of 3% of the total protein from each reaction is stored at —20° to provide the input reference sample in the subsequent analysis. 

Figure 9. Direct immobilization in PDMS of beads bearing biologically active compounds. Stepl Sepharose beads bearing bioactive compounds are arrayed as 0.3 pi drop onto a flat PVC master Step 2 a PDMS solution is poured onto the spotted array Step 3 the PDMS-based array is peeled off from the master and ready to use.

A flow assay was reported for determination of inorganic pyrophosphate a pyrophosphatase was coimmobilized with luciferase on Sepharose beads with continuous flow of saturating concentrations of substrates. The instrument allowed automation with a throughput of approximately one sample every 4 min. 

The sepharose beads were removed by brief centrifugation. 

Fig. 3. Fiow diagram of the expression and purification (a) and a representative resuit of the first preiiminary expression experiment (b) of GST-fused recombinant proteins. Fiow diagram (c left) and the representative resuit (c right) of the removai of GroEL directiy binding expressed GST-fused proteins on giutathione sepharose beads. The whoie ceii iysate of expressed bacteria and each wash and eiution fraction are ioaded on 12% SDS-PAGE (the numbers at the top of the panei are identicai to the numbers in the ieft diagram).

When the solubility assay gave an 5 value larger than 0.125, the GST-fiised protein extracted in a soluble fraction was purified on glutathione-sepharose beads. 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

Alterations in pH also can be responsible for the increase in solubility of loaded active agents. Glucose oxidase immobilized on sepharose beads were incorporated into ethyl vinyl acetate matrices along with insulin in the solid form. Glucose from blood enters these matrices, gets oxidized to glucuronic acid, and the decrease in pH increases the solubility of insulin, which diffuses out. The insulin in this case was modified by the addition of three extra lysine residues that ensured an isoelectric point of pH 7.4 for the molecule.33... 

Determine the efficiency of IgG immobilization to the Sepharose beads by measuring protein concentration in the supernatants obtained from steps 3. The protein remaining in these supernatant fractions represents antibody that was not immobilized. The efficiency of conjugation may be calculated by ... 

Efficiencies less than 70% indicate that inefficient IgG immobilization has occurred. The cause of inefficient coupling is often the presence of a buffer component (free amines in Tris-HCl buffers, for example) that competes with primary amines of the antibody for binding at the active sites of the cyanogen bromide activated Sepharose beads. 

A wide range of immobilization chemistries are commercially available in conjunction with Sepharose beads. We have investigated a limited subset of these possibilities which include direct, nonoriented immobilization via Schiff s base chemistry, oriented nonco-valent immobilization via immobilized metal affinity chromatography resins and oriented noncovalent immobilization via biotin-streptavidin binding. At present we favor direct, covalent attachment of proteins via primary amines since it is highly efficient (typically better than 85% yield), minimizes leaching and provides the best NMR results (Figure 6.2). 

May detect other lipid kinases such as PI 4-kinases that coassociate with immunoprecipitated proteins or nonspecifically associate with protein A-Sepharose beads. 

Fig. 1. Chemokines have different relative affinities for heparin in an inunobilized-heparin competitive binding assay. Radiolabeled chemokines were incubated with either Heparin-Sepharose beads (O), Sepharose beads (A), or without beads ( ) in the presence of increasing concentrations of heparin. Competition assays with MCP-1 were performed as described in the Methods section. Each point represents the mean S.E.M. of triplicate determinations, and the experiment shown is a representative of 2-A experiments for each chemokine. The maximal binding was 3000-5000 cpm, which corresponded to 15-20% of the total added radioactivity.

Immunoprecipitation Starter Kit, containing Protein A and Protein G Sepharose beads (GE Healthcare)... 

Fig. 2 Nucleic acid immobilization, (a) Immobilization of 5 -amino-C6 modified probes onto amino modified beads, (b) Grafting of biotinylated probes after the immobilization of avidin via reductive amination onto sepharose beads (3 xm)...

Fig. 3 Scanning electron microscopy (SEM) images of (a) a 150 pm-diameter latex beads spot, (b) a closer view of the latex beads arrangement within a spot (reproduced from [13]) (with permission) (c) immobilized streptavidine-coated beads (2.8 jim) on a biotin modified surface (reproduced from [14]) (with permission), (d) 3D representation of the SEM image of a Sepharose bead trapped at the PDMS/air interface (reproduced from [15]) (with permission)...

In another study, 5 -amino-C6 modified nucleic acid was immobilized onto cyanogen bromide activated Sepharose beads (Fig. 4B) [15]. The 100 pm-diameter beads were then subsequently transferred at the PDMS/air interface (Fig. 3D). 

Such porous polymeric beads have enabled a high enhancement of the specific surface since targets could be hybridized with the probes immobilized outside but also inside the Sepharose beads. 

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