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纯化资料

点击次数：  更新时间：2016/11/22 11:13:47  

Purification proteins

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