Dialysis tubing washing

Dialysis tubing (from Sigma Cat. No. D9652), flat width 33 mm, diameter 21 mm, cellulose (retains > 90% cytochrome c, MM 12.4 kDa, in solution over a 10 h period supplied in rolls, dry may contain glycerol and sulfur compounds in more than trace amounts washing instructions supplied). 

Activator-contain g fractions are combined and loaded onto a small (1-ml) column of octyl-Sepharose. Ampholytes are washed out with 5 ml of 10 mM phosphate buffer, pH 7 then the activator is eluted with 1% cholic acid (analytical grade) in the same buffer. After addition of 0.1 mg cytochrome c/ml (as protection against adsorption of the small amounts of protein onto dialysis tubing and other surfaces) the sample is dialyzed extensively against distilled water and then lyophilized. 

Transfer supernatant to dialysis tubing (boiled in EDTA and washed extensively in sterile water), and dialyze at d C against 3L of sterile O.lx DMEM, pH 4.0, without bicarbonate. Dialyze for three days, changing the medium once a day. Dialysis removes excess add while keeping the pH low (the collagen gels when alkahnized). 

Wash prepared dialysis tubing inside and out with sterile distilled water and then with high-salt conjugation buffer. 

Subject the solution obtained to dialysis and treat the washing water first with phenolphthalein and then with methyl orange. Pour the solution into a number of test tubes and add to them 1 N solutions of salts of mono-, di-, and trivalent cations, respectively. What is observed Explain the occurring processes. 

Affinity purification Dilute serum 1 10 with 10 mM Na-HEPES pH 8.0, centrifuge at 20,000 rpm for 30 minutes (Sorvall, SS34), and slowly load the supernatant onto the column. Afterward, wash extensively with 10 mM Na-HEPES pH 8.0 and 10 mM Na-HEPES pH 8.0, 300 mM KCl (at least 10 column volumes each). Some also wash with 200 mM NaSCN pH 5.8. Acid-sensitive antibodies are eluded with 100 mM glycine-HCl pH 2.5 (about 5 column volumes). Afterward, wash with 10 mM Na-HEPES pH 8.0 until the pH in the eluate is 8.0. Then, elude with 100 mM triethylamine pH 11.5 for the alkalinity-sensitive antibodies. Again, wash the column with Na-HEPES pH 8.0 and then elude with 3 M MgCl2. Start with a strong buffer in the fraction tube when you elude with pH extremes (e.g., 1/10 fraction volume 1 M Tris-Cl pH 8.1). In the end, adjust the column to pH 8.0 with 10 mM Na-HEPES, check the pH of the eluates (neutralize when required), and remove the MgCl2 (e.g., via dialysis). 

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