原核表达的His标签重组蛋白纯化范例－－艾莫瑞大学

endosome

Purifying Full-Length His-Tagged Importin α

1.        Obtain DE3 cells with full-length His-tagged importin α plasmid.
2.        Inoculate 4x5ml LB-Amp cultures.  Incubate at 37°C o/n.
3.        Inoculate 4L of LB-Amp with the o/n cultures.  Use 4ml of o/n culture for each 1L.  Incubate at 37°C for ~3hr until OD600=0.4-0.6 (Use 1ml LB as a blank; read 1ml of each culture).
4.        To induce, add 200 μl 1M IPTG (in box in -20°C freezer) to each 1L culture.
5.        Incubate at 30°C for ~5hrs.
6.        Transfer cultures to large white plastic spin bottles.  Spin down in centrifuge by eagle eye for 20min at 3,800rpm.  Dump the supernatant.
7.        Freeze in large plastic spin bottle at -80°C.
8.        Thaw on ice.  Resuspend in LB and combine pellets into a 50ml conical.  Spin 20min at 3,800rpm at 4°C.  Dump the supernatant.  Freeze pellet at -80°C, if necessary.  Thaw on ice.
9.        Resuspend pellet in Lysis Buffer.  Use an amount equal to the size of the pellet (ex. for 10ml pellet, use 10ml Lysis Buffer).
10.        French Press cells twice.
11.        Add DNase (amount depends on the volume of lysed cells).  Mix and leave at room temperature for 30min.
12.        Transfer to white spin tubes and centrifuge at 15,000rpm for 30min at 4°C.  Save supernatant in 50ml conical tube.  Put on ice.
13.        Obtain nickel column.  Notes for dealing with column:  Never let the column get air in it.  Therefore, for each wash, fill the neck of the column with water, attach the adaptor, fill the neck of the adaptor with water, fill the syringe, flick out all air bubbles, attach the syringe, and inject the solution at a pace where it slowly drips. Keep the column upright.
14.        Turn on lamp.  Turn on computer.  Make Stripping Solution, Nickel Sulfate, Buffer A, Buffer B, and Wash Buffer.  Set up tubes (~70).  Move the arm so that its mark is just in front of the first tube.
15.        Strip the column (removes any left-over proteins).
a.        Apply 2 bed volumes (10ml) of dH2O.
b.        Apply 2 bed volumes (10ml) of Stripping Solution--column should turn white.
c.        Apply 2 bed volumes (10ml) of dH2O.
16.        Charge the column--column should turn blue.
a.        For an old column, apply 14ml of 0.1M Nickel Sulfate.
b.        For a new column, apply 7ml of 0.1M Nickel Sulfate.
17.        Rinse column.
a.        For an old column, apply 10ml Buffer A.
b.        For a new column, apply 5ml Buffer A.
18.         Install column in cold cabinet.
a.        Unscrew adaptor.
b.        Clamp column.
c.        Attach bottom drain tube.
d.        Fill top of column with Buffer A and attach top feeder tube.
e.        Put paper towels underneath to see if the column is dripping.
19.        Open FPLC Director.  Go to Run/Control.
20.        Rinse system with water.
a.        Put Buffer A and Buffer B feeder tubes in dH2O.
b.        Manual→Pump→Wash→Waste→Check A and B→Start Wash
21.        Wash system with buffers.  
a.        Put Buffer A tube in Buffer A.
b.        Put Buffer B tube in Buffer B.
c.        Manual→Pump→Wash→Waste→Check A and B→Start Wash
22.        Rinse loop.
a.        Manual→Flow Path→Valves→New Position-Load→Set→Exit
b.        Fill syringe with needle with Wash Buffer.  Knock out bubbles.  Inject into little hole.  Should fill loop.  
c.        Repeat with Buffer A.  Leave syringe in.
23.         Wash column with Buffer A.
a.        Manual→Pump→Flow A+B→2ml→OK
b.        Run until blue UV280 line goes down (~10min).
24.        Auto zero.  Press button until red light comes on.
25.        Draw ~7ml of supernatant into syringe with needle.  Remove bubbles and inject into little hole.  Leave syringe in.
26.        Run Nickel1 program.  Wait for blue line to go down.  Keep the seven tubes that are filled in case the protein accidentally ran through.  Replace the seven tubes and reset the carousel.
27.        Detach column.  Wash the column with 2 volumes (10ml) Wash Buffer.
28.        Attach column.  Wash column on machine.  (Gets rid of Triton in Wash Buffer).
a.        Manual→Pump→Flow A+B→2ml→OK
29.        Run Nickel3 program.  Name the run.
a.        Will get two peaks.  Second is importin α.
b.        Run until conc.=100%B.
c.        Push End.  Save partial run.  Save all samples to run on acrylamide gel.
30.        Wash column on machine.
a.        Manual→Pump→Flow A+B→2ml→OK
31.        Go to Main Menu (at bottom of screen).  Evaluate which tubes have importin α by looking at chrom1 for the file.
32.        Run appropriate samples and 1 random sample and 1 sample of the crap.
a.        15μl sample + 3μl 5X RSB.  Boil at 100°C for 5min.  Run 10μl.
b.        Stain with Coomassie Blue for ~25min.  Destain and rinse with dH2O.
c.        Dry gel to save it.  Use cellulose membrane.  No bubbles.  Cut to size of drying apparatus, soak in dH2O, place bottom layer on, place gel on, place top layer on, clamp, and leave to dry on bench.
33.        Repeat steps 22-28 for the rest of the supernatant.
34.        Pool tubes that look good (those that are at the peak of importin α) into a 50ml conical on ice.  Discard other tubes.
35.        Dialyze.  (Gets rid of imidazole in sample).
a.        Cut ~9in of dialysis tubing (in cold cabinet—keep moist).
b.        Soak in dH2O for a few seconds.
c.        Clamp on one end.
d.        Fill with sample.
e.        Clamp top ~1in from the top of the sample.
f.        Put in 4L PBS + DTT at 4°C overnight with slow spinning.
36.        Turn lamp off.
37.        Strip the column (can be done the next day).
a.        Apply 2 bed volumes (10ml) of dH2O.
b.        Apply 2 bed volumes (10ml) of Stripping Solution--column should turn white.
c.        Apply 2 bed volumes (10ml) of dH2O.
d.        Apply 2 bed volumes (10ml) of Buffer A.
e.        Put column back in cold cabinet for use the next day. If must be stored, skip d, wash again with dH2O, and store in original box in 20% ethanol.
38.        Wash system with water (can be done next day).
a.        Put Buffer A and Buffer B feeder tubes in dH2O.
b.        ManualàPumpàWashàWasteàCheck A and BàStart Wash
39.        Wash system with ethanol (can be done the next day).
a.        Put Buffer A and Buffer B feeder tubes in 20% Ethanol.
b.        Manual→Pump→Wash→Waste→Check A and B→Start Wash
40.        Concentrate protein.
a.        Pour dialyzed protein into a 50ml conical on ice.
b.        Get concentrator (in microsope room—Millipore Amicon Ultra).
c.        Fill inside container to line with protein sample.  Screw on top.
d.        Spin at 3,000rpm for 10min at 4°C.  (Small things will cross filter and go into the exterior of the chamber).
e.        Discard the liquid in the exterior chamber.
f.        Repeat as needed, adding liquid to the interior chamber and spinning until sample is gone.  Do not concentrate too much or protein will precipitate.
41.        Bradford Assay
a.        Samples to read
i.        Blank (800μl dH2O + 200μl BioRad)
ii.        BSA Standards (800μl dH2O + 2, 4, 6, or 8μl 1:10 BSA + 200μl BioRad)
iii.        Importin α samples (800μl dH2O + 2 or 4μl sample + 200μl BioRad)
b.        Put 800μl dH2O in cuvettes.
c.        Dilute BSA 1:10 to a final concentration of 1mg/1ml.
d.        Pipette BSA into cuvettes.  Do not change tip.
e.        Pipette samples into cuvettes.  Do not change tip.
f.        Vortex cuvettes.
g.        Add 200μl of BioRad Protein Assay Reagent.  Vortex.
h.        Measure OD at 595.
i.        Use Cricket Graph to find concentration.
i.        Enter data for standards
ii.        Graph→New Graph→Line    y=mg/ml    x=OD
iii.        Options→Curve Fit→method(linear)    .98<r<1
iv.        Use equation to calculate concentration of sample.  Plug in the average of the readings for the sample for x.  y=conc.
42.        If concentration is too low, concentrate again.
a.        May have to use small concentrator.  
b.        Apply liquid to the top.
c.        Spin at 3,000rpm for 30min at 4°C.
d.        Keep liquid on top.  Use a transfer pipette to squirt the membrane and free protein.  Save.
e.        Repeat as necessary.  Find concentration.
43.        Add 10% glycerol, aliquot, store at -80°C.

Materials

1M IPTG   (check for aliquots in -20°C freezer first)
        23.83g IPTG
        100ml dH2O

Lysis Buffer        to make 50ml
        20mM Tris-HCl pH 7.8        5ml of 200mM
        2mM β-Mercaptoethanol        6.99μl
        2mM PMSF        1ml of 0.1M
        2mM EDTA        200μl of 0.5M
        0.1% Triton X-100        50μl of 0.1%

        200mM Tris-HCl pH 7.8
                6.057g Tris Base Ultrapure
                250 ml dH2O
                                -pH to 7.8 with HCl
                                -Filter sterilize

0.1M PMSF
        0.871g PMSF
        50ml ethanol

0.5M EDTA (check stocks of solutions first)
        186.0g EDTA
        1L dH2O
                -pH to 8.0 with NaOH
                -Filter sterilize

DNase                per 1ml lysed cells        for 16ml of lysed cells
        120 Unit Dnase (-20°C)        16.6μl        ~400μl
        1M MnCl2        2.4μl        38.4μl
        1M MgSO4        24μμl        384μl

        1M MnCl2
                9.90g MnCl2
                50ml dH2O
                        -filter sterilize

        1M MgSO4
                12.33g MgSO4
                50ml dH2O
                        -filter sterilize



Stripping Solution
        5.844g NaCl
        3.722g EDTA
        100ml dH2O

0.1M Nickel Sulfate
        1.314g Nickel Sulfate
        50ml dH2O        

Buffer A          for 800ml
        76ml 0.1M Sodium Phosphate Monobasic
        324ml 0.1M Sodium Phosphate Dibasic
        11.688g NaCl
        400ml dH2O

        0.1M Sodium Phosphate Monobasic
                12.0g Sodium Phosphate Monobasic
                1L dH2O

        0.1M Sodium Phosphate Dibasic
                14.2g Sodium Phosphate Dibasic
                1L dH2O

Buffer B
        800ml Buffer A
        27.232g Imidazole

Wash Buffer
        499.5ml Buffer A
        0.5ml Triton-X-100

PBS + DTT
        400ml 10x PBS
        1.23g DTT
        3600ml dH2O

        10x PBS
                80g NaCl
                2g KCl
                21.8g Na2HPO4·7H2O
                                -add dH2O to a final volume of 1L
                                -pH 7.2-7.4
                        -Filter sterilize

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