Prepare Column Resin

· Wash resin with TSA (5 x 10 ml)

· Resuspend resin in 2 ml final vol TSA

Prepare immune serum.

· Spin serum (IX-63 IS 7.3, 1/7/00) for 1 min at 16,000 g to pellet any insoluble material.

· Dilute 1 ml IS 7.3 with 9 ml TSA

Purify specific antibodies.

· Add diluted immune serum to column resin.

Rock at Room Temp for 3-4 hr

TIME ON: 4:30 pm

TIME OFF: 8:00 pm

----Save ALL volumes obtained below!!----

· Spin for 2 min setting #3 clinical centrifuge (300 – 700 xg)

supe = "FT-1"

· Wash resin with 5 ml TSA

supe = "FT-2"

· Add 5 ml TSA to resin and pour column, rinse with 5 ml more TSA

flow through = "FT-3"

· Wash with 10 column volumes TSA (5 ml)

flow through = "FT-4"

· Wash with 10 volumes TBS/Azide Wash Buffer "W1"

· Wash with 10 vol High Salt TBS-Triton pH8 ("W2")

· Wash with 10 vol High Salt TBS-Triton pH9 ("W3")

· Wash with 10 vol High Salt TBS-Triton pH8 ("W4")

· Wash with 10 vol High Salt PBS-Triton pH6.5 ("W5")

--changes in pH are done to release additional non-specifically bound proteins—

· ELUTE with 2.5 ml (5 col volumes) Glycine/NaCl/Triton, pH 2.5

Collect 25 x 0.1 ml fractions CONTAINING 0.02 ml 1 M Tris pH9—the Tris is essential for neutralizing the sample so as to avoid unnecessary damage to antibodies.

· Store all fractions at 4 deg C.

· Wash with another 2.5 ml Glycine buffer ("E26")

· Rinse column with 20 vol (10 ml) TSA ("Recharge")

· Store column at 4 deg.

· ----For long term column storage, add preservatives to TSA

1 mM EDTA + 20 ug/ml gentamycin

or

0.01 % thimerosal