Chromatography buffer

Immediately quench the reaction by the addition of sodium sulfite (Na2S03) to provide a 2X molar excess over the initial amount of periodate added. Purify the oxidized antibody by gel filtration using a desalting resin. The chromatography buffer is 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2. To obtain efficient separation between the oxidized antibody and excess periodate, the sample size applied to the column should be at a ratio of no more than 5 percent sample volume to the total column volume. Collect 0.5 ml fractions and monitor for protein at 280 nm. 

E. Merck cellulose thin-layer chromatography plates (available from American Scientific Products) are developed with chromatography buffer (Note 13) and visualized with sulfosalicylic acid/ferric chloride spray. The system consists of a solution of 1.0 g of sulfosal icyl ic acid (from Aldrich... 

One half of the concentrated extract, dissolved in an equal volume of chromatography buffer (Note 13), is loaded onto a 5. 5 x 18-cm cellulose flash... 

Chromatography buffer is prepared by dissolving 4.0 g of ammonium bicarbonate in 250 mL of deionized water and adding 500 mL of isopropyl alcohol and 250 mL of acetonitrile. The resulting solution is approximately 50 mM in ammonium bicarbonate. 

Immediately purify the maleimide-activated HRP away from excess cross-linker and reaction by-products by gel filtration on a desalting column (Sephadex G-25 or the equivalent). Use 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, as the chromatography buffer. HRP can be observed visually as it flows through the column due to the color of its heme ring. Pool the fractions containing the HRP peak. After elution, adjust the HRP concentration to 10 mg/ml for the conjuga-... 

Cation exchange chromatography buffers Buffer A 50 mM sodium acetate, pH 4.5. Buffer B Buffer A containing 2 M NaCl. 

Hydrophobic interaction chromatography buffer Buffer A 50 mM sodium phosphate, pH 7.0, containing 1.7 M ammonium sulfate (see Note 4). Buffer B 50 mM sodium phosphate, pH 7.0. 

S. Espinosa, E. Bosch, and M. Roses, Retention of ionizable compounds on HPLC. 12. The properties of liquid chromatography buffers in acetonitrile-water mobile phases that influence HPLC retention. Anal. Chem. 74 (2002), 3809-3818. 

The sample is loaded on the paper as described for paper electrophoresis but the paper is not sprayed before the run ( 3.1.3). The bottom edge of the paper is serrated to allow buffer to drip off evenly and the paper is loaded, dry, into the chromatography tank. The atmosphere in the tank and the paper are allowed to equilibrate with a beaker of the chromatography buffer placed in the tank for at least 0.5 hr. Then the upper trough (for descending chromatography) is filled with the buffer and chromatography commences. 

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