纯化资料
丁香园论坛
2005
Purification proteins by TALON
50 ml Cells weight (mg) Bugbuster (ml) 1´Extraction buffer (ml) TALON (ml) Resin (ml) Elution buffer (ml)
300 2.0 2.0 1000 500 250
2.0 ml eppendorf tubes should be used for mini-purification. For this purpose, if the total volume is more than 2 ml, try to use 5´Extraction buffer instead of 1´Extraction buffer.
5´Extraction buffer: 1.0 ml supernatant + 250 ml 5´Extraction buffer
1.5 ml supernatant + 375 ml 5´Extraction buffer
check buffer pH just before using
this protocol may need to be optimize for diffirent proteins
Day 1.
Sample preparition:
1. Weight
2. add Bugbuster (Benzonase to be added)
3. resuspend it by inverting
4. incubate 20 min at RT.
5. centrifuge 20 min at 4°C, 13000rpm
6. supernatant (soluble proteins)
take 40 ml out for SDS-PAGE checking (as "before purification")
to the left, add 1´ or 5´ native extraction/wash buffer pH 7.0, this is clarified sample, ready for combining with TALON.
7. Pellet (insoluble proteins)
Add 1´ denaturing extraction/wash buffer pH 7.0
resuspend it
agitate 20 min at RT (or until it become translucent),
centrifuge 15 min at 4°C, 13000 rpm,
transfer the supernatant to a new tube (this is clarified sample)
take 40 ul out for SDS-PAGE checking (as "before purification").
Column preparition(pre-equilibrate):
1. take appropriate amount volume of TALON
2. centrifuge 2 min at 700 rcf (g), throw away supernatant
3. add 1 ml 1´ extraction buffer (native or denaturing)
4. centrifuge 2 min at 700 rcf, throw away supernatant
5. repeat once.
Binding:
1. add clarified sample to the resin
2. agitate 20 min at RT
3. centrifuge 5 min at 700 rcf
4. take supernatant out, keep it (as "no bands" fraction)
Washing (2´):
1. add 1.5 ml 1´ extraction buffer
2. agitate 10 min at RT
3. centrifuge 5 min at 700 rcf, take supernatant out, keep it (as "wash 1")
4. repeat once ("wash 2")
Elute (pre-elute+elute):
1. add 1.5 ml 1´ extraction/wash buffer to the resin
2. transfer it to the pre-equilibrated column
3. allow the resin settle down
4. remove the end-cap, allow the buffer to drain
5. pre-elute the column with pre-elute buffer for 5 times, keep each fraction
6. elute with elution buffer for 8 times, keep each fraction
SDS-PAGE:
1. 7 ml of proteins +7 ml sample buffer
2. load 10 ml on gel
3. 10 ul prestained protein marker
Dialysis:
1. transfer fractions containing proteins into membrane , tie each end, dialysis in 2 L PBS at 4°C o/n.
Day 2.
1. Fresh PBS, dialysis another 2 h (at least)
2. aliquote proteins, 20°C store it (at least 100 mg required for phage selection).
SDS-PAGE:
1. 7 ul of proteins +7 ul sample buffer, load 10 ul on gel
2. load 10 ul of 10, 50, 100, 500 ug/ml BSA (7:7 BSA: sample buffer) on gel for checking protein concentration
3. 10 ul prestained protein marker
50 ml Cells weight (mg) Bugbuster (ml) 1´Extraction buffer (ml) TALON (ml) Resin (ml) Elution buffer (ml)
300 2.0 2.0 1000 500 250
2.0 ml eppendorf tubes should be used for mini-purification. For this purpose, if the total volume is more than 2 ml, try to use 5´Extraction buffer instead of 1´Extraction buffer.
5´Extraction buffer: 1.0 ml supernatant + 250 ml 5´Extraction buffer
1.5 ml supernatant + 375 ml 5´Extraction buffer
check buffer pH just before using
this protocol may need to be optimize for diffirent proteins
Day 1.
Sample preparition:
1. Weight
2. add Bugbuster (Benzonase to be added)
3. resuspend it by inverting
4. incubate 20 min at RT.
5. centrifuge 20 min at 4°C, 13000rpm
6. supernatant (soluble proteins)
take 40 ml out for SDS-PAGE checking (as "before purification")
to the left, add 1´ or 5´ native extraction/wash buffer pH 7.0, this is clarified sample, ready for combining with TALON.
7. Pellet (insoluble proteins)
Add 1´ denaturing extraction/wash buffer pH 7.0
resuspend it
agitate 20 min at RT (or until it become translucent),
centrifuge 15 min at 4°C, 13000 rpm,
transfer the supernatant to a new tube (this is clarified sample)
take 40 ul out for SDS-PAGE checking (as "before purification").
Column preparition(pre-equilibrate):
1. take appropriate amount volume of TALON
2. centrifuge 2 min at 700 rcf (g), throw away supernatant
3. add 1 ml 1´ extraction buffer (native or denaturing)
4. centrifuge 2 min at 700 rcf, throw away supernatant
5. repeat once.
Binding:
1. add clarified sample to the resin
2. agitate 20 min at RT
3. centrifuge 5 min at 700 rcf
4. take supernatant out, keep it (as "no bands" fraction)
Washing (2´):
1. add 1.5 ml 1´ extraction buffer
2. agitate 10 min at RT
3. centrifuge 5 min at 700 rcf, take supernatant out, keep it (as "wash 1")
4. repeat once ("wash 2")
Elute (pre-elute+elute):
1. add 1.5 ml 1´ extraction/wash buffer to the resin
2. transfer it to the pre-equilibrated column
3. allow the resin settle down
4. remove the end-cap, allow the buffer to drain
5. pre-elute the column with pre-elute buffer for 5 times, keep each fraction
6. elute with elution buffer for 8 times, keep each fraction
SDS-PAGE:
1. 7 ml of proteins +7 ml sample buffer
2. load 10 ml on gel
3. 10 ul prestained protein marker
Dialysis:
1. transfer fractions containing proteins into membrane , tie each end, dialysis in 2 L PBS at 4°C o/n.
Day 2.
1. Fresh PBS, dialysis another 2 h (at least)
2. aliquote proteins, 20°C store it (at least 100 mg required for phage selection).
SDS-PAGE:
1. 7 ul of proteins +7 ul sample buffer, load 10 ul on gel
2. load 10 ul of 10, 50, 100, 500 ug/ml BSA (7:7 BSA: sample buffer) on gel for checking protein concentration
3. 10 ul prestained protein marker
