Binding buffers

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. 

Apply the conjugate to the column in the binding buffer while taking 2 ml fractions. 

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

Wash the column with 10 volumes of water, then equilibrate the support with 2 volumes of 10 mM sodium phosphate, 0.15 M NaCl, pH 7.0 (binding buffer). 

Dissolve or dialyze the conjugate into binding buffer. Apply the conjugate solution to the column while collecting 2ml fractions. 

Interactions between proteins and salts in the binding buffer are also a major determinant of selectivity. Salts that are strong retention promoters in HIC are excluded from protein surfaces by repulsion from their hydrophobic amide backbones and hydrophobic amino acid residues.8,9 This causes the mobile phase to exert an exclusionary pressure that favors the association of proteins with the column, regardless of stationary-phase hydrophobicity.1(W2 Because this mechanism involves the entire protein surface, the degree of exclusion is proportional to average protein hydrophobicity, regardless of the distribution of hydrophobic sites. 

Table 6.1 Buffers for Initial Selectivity Screening pH 8.5, binding buffer...

Note also that ammonium sulfate is absent from the recommended binding buffer formulations despite its general popularity in the field. Ammonium ions become fully titrated at alkaline pH and convert to ammonia gas. Buffer pH may become unstable as a result and causticity of the free ammonia may partially hydrolyze the proteins in a sample, creating a source of assay variability.617 18 At small buffer volumes used for analytical applications, liberated ammonia gas may not be a significant health hazard, but precautions may still be necessary to meet regulations. For all these reasons, ammonium salts are best avoided at alkaline pH. 

Table 6.2 Separation Conditions for Initial Screening Equilibrate column 5 column volumes (CV) 100% binding buffer3 Inject sample 2% CV unequilibrated sample6 Wash 2 CV binding buffer...

In order to select a carrier solution composition which would provide an overall maximum response for MS detection, two modifiers were selected, acetonitrile and methanol, and two buffers, i.e. ammonium acetate (10 mmol pH 7.5) and ammonium formate (10 mmol L pH 7.5). Biotin and fluorescein-biotin were dissolved in various binding buffer-organic solvent mixtures ranging from 90 10 (v/v) to 50 50 (v/v) at two concentration levels (0.01 ng 1 ng pL ) and 20 pL were injected and analyzed by MS in full-scan and SIM mode. The maximum response was found with 50% methanol, which was about a factor 2x higher than for 10% methanol. Since the proteins can denaturate or protein-ligand complexes can dissociate at relatively low percentages of organic modifier in further experiments only 10% methanol is used in the carrier solution. 

To this end, the pellets remaining from the competitive MS binding assay were, after several washing steps, resuspended in binding buffer and incubated with a great excess of competitor (50 pM (+)-methadone) to liberate the unknown bound ligand (as well as the bound marker). Then the supernatants obtained by centrifugation were analyzed by LC-ESl-MS/MS. In addition to morphine as the marker, naloxone was identified as the hit that had been searched for. Thereby, the relative concentrations of marker (2.93 nM) and hit (2.30 nM) pointed to the fact that the hit had a similar affinity to the //-opioid receptor as the marker [65]. 

Add the cell lysate to a prepacked MIcroSpIn GST or GSTrap FF column equilibrated with the binding buffer. 

Equilibrate the column with 5 column volumes of the binding buffer. 

Wash with 5-10 column volumes of binding buffer. 

Add 0.5 -1 ml of protein sample, dialyzed against the respective buffer, to the wet media. Incubate for 15 - 30 min at that temperature which should be used for separation. Separate by centrifugation or filtration after incubation and determine the amount of target protein in the liquid. For binding buffer select that buffer at which the target protein disappears. 

The capacity of the exchange medium is determined with a series of protein dilution in binding buffer and a constant amount of lEC gel. 

Mix the protein dissolved in binding buffer with equilibrated lEC gel. Agitate for several minutes and separate gel and solution either by centrifugation or filtration. Wash and elute the gel on a funnel or in a column. Since the equilibrium is established only once, a nearly complete absorption occurs if the gel is in a large excess. The advantage of batch loading is the easy separation of gel and liquid. 

It is possible to combine stepwise gradient and continuous gradient, i.e., the starting elution buffer has a higher ionic strength than the binding buffer, and after a short time the elution of the protein(s) of interest starts by application of a flat continuous gradient. 

Affinity medium is equilibrated with binding buffer... 

Pack 4 mL of the gel in a polypropylene column, and equilibrate with 25 mL of binding buffer (0 lMTris-HCl buffer, pH 7.6, containing 0.5MK2SO4)... 

After the sample has entered the gel, wash non-IgG from the column with 20 mL of binding buffer monitor the A280 as an indicator of protein content in the wash until the absorbance returns to background levels... 

Perform the chromatography at 4°C Equilibrate the column with 10 mM Tns-HC1 buffer, pH 7 4, containing 65 mM NaCl (binding buffer). 

When the sample has entered the column matrix, wash with binding buffer until the absorbance falls to baseline (<0 02). 

Following dialysis, mix the ascitic fluid with an equal volume of binding buffer, and apply to the column... 

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