Plasma AAT prep
Buffers
Saturated ammonium sulfate with 0.28% v/v BME
50% saturated ammonium sulfate with 0.14% v/v BME
Dialysis Buffer
5mM EDTA, pH 8.0
Equilibration Buffer
0.1M Tris, 5mM EDTA, pH 8.0
DTNB Solution
0.1% w/v 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB), 0.1M Tris, 5mM EDTA, pH 8.0
DTNB/DTT Solution
40mg DTNB plus 8mg DTT in 40ml Equilibration buffer
Concentrating Buffer
0.1M Tris, 5mM EDTA, 0.1% v/v BME
Buffer QA
0.1M Tris, 5mM EDTA, pH 8.6
Buffer QB
0.1m Tris, 5mM EDTA, 0.4M NaCl, pH 8.6
Storage Buffer
0.05M Tris, 0.05mM KCl, 0.2% BME
Day 1
Make up 300ml of 100% saturated ammonium sulfate and allow to mix overnight
Day 2
Defrost 1 bag of plasma at 37C in a waterbath until completely liquid
Add 0.28% v/v BME to the ammonium sulfate, then mix with equal amount of plasma and stir at 4C
for 15 min
Centrifuge the mixture at 6000rpm for 20 min, 4C, and retain the supernatant in a separate beaker
Resuspend the precipitate with 200ml of 50% saturated ammonium sulfate containing 0.14% v/v
BME.
Centrifuge at 8000rpm for 20 min at 4C and retain supernatant
Mix the supernatant from the previous steps and add solid ammonium sulfate to the mixture such that
the final solution is at 70% saturation.
Adjust to pH 6.0 with H2SO4, then stir gently for 30 min at 4C.
Centrifuge mixture at 10,000rpm for 45 min at 4C.
Pour off supernatant and resuspend the pellet in a minimum volume of equilibration buffer (< 150ml).
Dialysis 1: protein placed in dialysis bags and dialysed in 5L of Dialysis Buffer (5mM EDTA, pH 8)
for 2 hrs at 4C.
Dialysis 2: Change to 5L of fresh Dialysis buffer and leave for 4 hrs at 4C.
Dialysis 3: Change again to fresh 5L of dialysis buffer and leave overnight at 4C.
Day 3
Wash glutathione resin with ~200ml of equilibration buffer at 4 ml/min.
Charge column by adding 50ml of DTNB solution at 4 ml/min.
Wash column again with ~200ml of equilibration buffer at 4 ml/min.
Load protein solution from dialysis at 4 ml/min and collect all flow through, all at 4C.
Wash column with ~130ml of equilibration buffer containing 0.5M NaCl at 4 ml/min, collecting the
flow through.
Wash further with ~200ml equilibration buffer alone at 4 ml/min, flow through not collected.
Elute protein by adding 40ml of FRESH DTNB/DTT solution to the column at 4 ml/min, collecting
10ml fractions.
Repeat this process again: Wash glutathione resin with ~200ml of equilibration buffer at 4 ml/min.
Charge column by adding 50ml of DTNB solution at 4 ml/min.
Wash column again with ~200ml of equilibration buffer at 4 ml/min.
Load protein solution from dialysis at 4 ml/min and collect all flow through, all at 4C.
Wash column with ~130ml of equilibration buffer containing 0.5M NaCl at 4 ml/min, collecting the
flow through.
Wash further with ~200ml equilibration buffer alone at 4 ml/min, flow through not collected.
Elute protein by adding 40ml of FRESH DTNB/DTT solution to the column at 4 ml/min, collecting
10ml fractions.
Day 4
Repeat again another time: Wash glutathione resin with ~200ml of equilibration buffer at 4 ml/min.
Charge column by adding 50ml of DTNB solution at 4 ml/min.
Wash column again with ~200ml of equilibration buffer at 4 ml/min.
Load protein solution from dialysis at 4 ml/min and collect all flow through, all at 4C.
Wash column with ~130ml of equilibration buffer containing 0.5M NaCl at 4 ml/min, collecting the
flow through.
Wash further with ~200ml equilibration buffer alone at 4 ml/min, flow through not collected.
Elute protein by adding 40ml of FRESH DTNB/DTT solution to the column at 4 ml/min, collecting
10ml fractions.
Protein detection: using Bradford’s reagent, mix 20uL of protein from each fraction with 80uL of
reagent in a plate. Coloured fractions contain protein.
Concentrate protein: pool all fractions containing protein and concentrate in the Amicon concentrator
through 2 cycles of 180ml20ml, changing the buffer as you go to concentrating buffer. Finally
dilute the protein out again to 50ml.
Equilibrate 50ml Q sepharose column with Buffer QA.
Load protein onto the column at 2ml/min, 4C.
Wash unbound protein with ~200ml of Buffer QA at 2ml/min until no more protein is coming off the
column.
Elute protein with a linear gradient of 400ml, 0-0.4M NaCl (mixing Buffers QA and QB) at 1ml/min.
Run this all at 4C overnight, allowing the column to dry out during this process.
Day 5
Check fractions for protein using Bradford’s reagent (protein previously seen in fractions 15-59).
Check activity of each fraction by mixing 20uL of various fractions with 20uL of 200nM chymo,
incubating at 37C for 15 min then adding diluted pNA and reading A405.
Run various samples on both native and SDS PAGE to confirm the presence of protein and that is it
monomeric, in each case 10uL of protein with 10uL loading dye is sufficient.
Pool fractions that have monomeric, active protein, and concentrate, changing the buffer over to
storage buffer.
Check protein concentration and do SI to confirm. Aliquot and store at -80C.