PROTOCOLS 1

B:WGALG.SAM 7/25/92

LARGE WGA PREP

A) GROW UP A LARGE (10 l) SPINNER FLASK OF CELLS (CHO-IR):

1. Clean the spinner flask by scrubbing with a brush (no detergent in TC flasks), or, if that doesn't do it, soak the flask overnight in 0.05 N NaOH, and scrub. Rinse in deionized water, and silanize the flask with Prosil-28 (PCR). Make a 1:100 dilution of Prosil-28 in deinoized water (Prosil is very toxic, use gloves and caution) and slosh it around the clean spinner so that all parts of the spinner get coated. Rinse the spinner twice with tap water and twice with deionized water. Air dry at least overnight. Autoclave the spinner with a 0.2 mM filter over one ventilation port.

2. Grow 10 150 mm plates of CHO-IR cells to confluency. Trysinize the cells, stop the trypsin with McCoy's 5A media containing 9% CS, 1% FBS, and spin out the cells. Resuspend the cells and add to 1 l of McCoy's 5A media with 9% CS, 1% FBS in an autoclaved spinner. Put the spinner in the incubator on a stir plate, adjust so that the spinner doesn't click, and hook up air pump to filter so that air/CO_{2 is recirculated sterilely through the flask.}

_{3. After 3 days check the cells daily, feed them to 5 liters when they get orange (same medium). At some point in time "ovaries" (large clumps of CHO cells) will appear in the spinner. When the media again turns orangish, and there are ovaries in the spinner, feed the flask to 10 liters. You will harvest the cells in 2-3 days.}

_{B) MAKE THE PREP}

_{1. Get 10 ml of WGA-sepharose (Pharmacia) in a column hooked to a peristaltic pump (in the cold room). If it has been previously used and you aren't sure of its state of cleanliness, wash with 100-200 ml of 0.1% SDS in deionized water, followed by 100-200 ml of deionized water, before equilibrating it with 100-200 ml of 0.1% Triton X-100 in 50 mM HEPES pH 7.4 (H/T).}

_{2. Spin down the cells in 1 liter bottles in Spiro's old IEC centrifuge. Since the centrifuge holds only 4 bottles, if takes three spins. Each spin is 25 minutes at 1000 rpm. After each spin, aspirate (do not pour- loose pellet) the media and add more cells from the spinner. When all of the cells are spun, aspirate all of the media off.}

_{3. Solubilize the cell pellet by resuspending it in 200 ml of ice cold 50 mM HEPES containing 1.0% Triton X-100, 2 mM PMSF, and 0.01 mg/ml Aprotinin. Stir on ice or in the cold room 45-60 minutes to break up clumps and solubilize the cells.}

_{4. Pellet crap out of the the lysate by spinning the lysate in a Ti35 rotor for 1 hour at 31 krpm.}

_{5. Load the column by pumping the lysate supernatant over the column overnight at 0.5 ml/min.}

_{6. Wash the column with 100-200 ml of H/T, and elute at a flow rate of 0.5 ml/minute with H/T containing 0.3 M N-Acetylglucosamine (6.6 mg/100 ml). Collect 20 3-4 ml fractions and assay for protein using Bio-Rad assay. Pool peak fractions usually 2 fractions between fractions 6-10.}

_{7. Freeze 100 ul fractions at -70}^o C.

8. Wash the colum with 0.1% SDS, deionized water and H/T as described above (B1). Store the column in H/T containing 0.02% sodium azide or repeat steps B5-B7 by reloading the effluent lysate. Second recovery is about 1/2 to 1/3 of first.