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GST pull down assay
A. Expression of GST fusion protein
1. Inoculate 2ml of LB/antibiotics with 1 colony
2. O/N culture with shaking at 37℃
3. Inoculate pre-warmed 25ml of LB/antibiotics with all of
2ml-cultrure
4. 1 hr at 37℃ with shaking vigorously
5. Inoculate pre-warmed 500 ml of LB/antibiotics with all of
25ml-culture
6. Shaking incubation at 37℃ until OD = 0.45
7. Add 0.1mM IPTG
8. 3 hrs at 30℃
l if insoluble protein, replace the step 6-8 with followings
6.shaking incubation at 37℃ until OD = 0.2
7.shaking incubation at 25℃ for 1 hr
8.add 0.1mM IPTG when OD=0.45
9. 3 hrs at 25℃ with vigorous shaking
10.Aliquote 10 – 15 ul of culture and spin down for expression test later
11.Harvest with centrifuge at 7000 – 8000 rpm, 4℃, 10 min
12. Wash with 10 ml of 1x PBS once
13. Go to the next step or – 70℃
B. Check the expression of fusion protein
1. wash the pellet(from A. step 10) with 1 ml of 1x PBS once
2. spin down with table-top centrifuge for 30 sec
3. resuspend the pellet with 100 – 200 ul of 1x PBS
4. sonicate : dial 10-11(= output power 50-60%)
10 sec and pause 30-60 sec in ice/ repeat 2-4 times
5. centrifuge 12000-14000 rpm for 1 min, RT or 4℃
6. take supernatent into new tube
7. resuspend the pellet with same volume of 1x PBS
8. add sample buffer to 15 ul of soup and resuspended pellet
9. boil for 5 min
10. electrophoresis
C. Isolation of fusion protein
1. prepare 1x extraction buffer ;
40 ul of 1M Hepes(pH7.5), 200 ul of glycerol, 1 ul of 1M DTT, 3 ul of 100uM of PMSF, 1ul of 1000x leupeptin, 1ul of 1000x pepstatin,10 ul of 2M NaCl, 4ul of 0.5M EDTA and finally adjust the volume with D.W. to 1ml
2. measure the mass of cell
3. add 5ml of extraction buffer/ g of cell mass
4. suspend the pellet completely, if needed use glass rod or tip
5. add 60 ul of lysozyme(50 mg/ml) and vortex
6. incubation on ice for 30 min
l if insoluble protein, add dropwise while vortexing 60 ul of 100% tween 20 and 60 ul of 100 % triton X-100, to a final concentration of 1% each.
7. sonicate sample for 15-20 sec and pause 30-60 sec on ice
repeat 3-4 times until the suspension is cleared
8. centrifuge at 15,000 rpm,4℃, 20min
9. aliquot the soup to 1ml per tube.
10. – 70℃
D. Purification of fusion protein
1. aliquot 40 ul of glutathione sepharose bead in a tube
2. wash the bead with 1 ml of 1x PBS
3. spin down for 7-10 sec with table-top centrifuge RT
4. remove the buffer as completely as possible
5. add 300 ul of 1x PBS and various amount of fusion protein
(usually 50-300 ul)
6. suspend tapping or vortexing briefly
7. rotate at rolling machine for 15 min at RT or 4℃
8. wash with 1ml of 1x PBS 2-3 times
(remove buffer as completely as possible after spindown)
9. add 40 ul of 15 mM reduced glutathione( in 50 mM Tris pH 8.0)
10. flick the tube or vortex briefly and incubate for 15 min RT or 4℃
11. spin down for 10-15 sec and carefully take 20 ul of soup
12. add 5 ul of 5x sample buffer and boil for 5 min
13. electrophoresis and stain/destain
E. Interaction test
1. prepare interaction buffer( same component as extraction buffer). To control binding, change salt concentration. For stronger band in the gel increase salt up to 150mM of KCl.
2. after washing at the step 8 of D, add 300ul of 1x interaction buffer with 10mg/ml BL21 crude extract (BL21 host without plasmid prepared by A)
3. add 2-3 ul of in vitro translated protein
4. rotate at 4℃ for 3-4 hours or O/N on rolling machine after tapping or vortexing briefly
5. spin down and remove buffer as completely as possible
6. wash with 1 ml of interaction buffer 3 times. If your protein is sticky, use 0.1- 1% triton X-100 at this washing step.
7. elute with 40 ul of 15mM reduced glutathione as before
8. spin down and carefully take 20 ul of soup and analyze on SDS-PAGE or autoradiography
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