Berglund:U2AF65

From OpenWetWare

Jump to: navigation, search

Materials

LYSIS BUFFER
25mM Tris pH 7.5
300mM NaCl
1mM EDTA
2mM DTT

GLUTATHIONE ELUTION BUFFER
50mM Tris pH 8
3.08mg/mL reduced glutathione
(may want to add salt for protein stabilization)

10X PRESCISSION PROTEASE CLEAVAGE BUFFER
500mM Tris pH 7
1.5M NaCl
10mM EDTA
10mM DTT
0.1% Triton X-100
(I no longer put DTT in this buffer, as I'm not sure it lasts with the buffer being stored at 4˚. Just add fresh DTT to cleavage reaction.)

S BUFFER A
10mM MES pH 6.5
100mM NaCl
1mM EDTA
filter with 0.22µm filter
2mM DTT

S BUFFER B
10mM MES pH 6.5
1M NaCl
1mM EDTA
filter with 0.22µm filter
2mM DTT

DIALYSIS/STORAGE BUFFER
25mM Tris pH 7.5
300mM NaCl
1mM DTT
15% glycerol

Methods

1. Start a 50mL overnight culture from frozen stock of E.coli carrying the protein expression plasmid. (Scrape a bit out of the frozen stock with a toothpick and drop toothpick into culture.)
a. 50mL 2X YT broth
b. appropriate antibiotics as needed
i. Ampicillin (2X) 100µg/mL
c. 37˚ overnight with shaking (~200-225rpm)

2. Scale-up: 1 - 2L
a. Fernbach flasks preferred
b. Per 1L 2X YT broth, add antibiotics as above
c. Inoculate 10-25mL of the overnight culture into each L (same volume for each)
d. Grow at 37˚ with shaking to OD600 of ~1.0
e. Take a 100µL sample of uninduced cells, spin down, retain cell pellet at -20˚

3. Induce protein expression using Lac promoter
a. Per liter of culture, add IPTG to final concentration of 0.25-1mM
b. Continue to incubate at 37˚ for 3 hours
c. Take 100µL sample of induced cells, save cell pellet at -20

4. Pellet cells: spin in giant rotor at 5000rpm for ten minutes. Pour off media, scrape cell pellets into a 50mL conical tube, and save pellet at -20˚ overnight.

5. Resuspend pellet in lysis buffer (up to 35-45mL on top of pellet in tube). This can be done on the rotator at 4˚C and/or with vortexing, etc.
a. If using already made buffer, filter anew and add fresh DTT to 2mM

6. Sonication: power setting 5-7, 3-5 x 30sec bursts
a. Transfer cell suspension to small beaker.
b. Keep on ice and check between bursts to keep from overheating.
c. Tune sonicator according to protocol

7. Pellet insoluble cell debris: spin in JA-17 rotor at 20,000xg (12,000rpm) for 30 minutes.
a. Sample: 3µL of extract (supernatant) + 12uL H2O -> -20
b. Sample: pellet (scrape a little into 12µL H2O) -> -20
c. Save pellet(s) at 4˚ until gel has been run

8. Prepare GST beads by washing with lysis buffer.
a. 7.5mL slurry glutathione sepharose beads (Amersham) (shake to resuspend) (5mL beads)
b. to 35-45mL with lysis buffer
c. spin in cooled clinical centrifuge (molbio centrifuge room) 1000rpm, 3min
d. Discard supernatant
e. Repeat for total of 3 washes

9. Bind protein to GST beads: add extract from step 7 to washed beads and incubate on rotator at 4˚ for 1 hour.

10. Collect unbound proteins by spinning in clinical centrifuge for 4min at 1000rpm. Tranfer supernatant (unbound) to new tube and save at 4˚ until gel has been run.
a. Wash beads 3x with lysis buffer.
b. Sample: 3µL unbound + 12uL H2O
c. Sample: 6uL bound beads + 6uL H2O

11. Elute fusion protein by competition with reduced glutathione
a. Add 10mL elution buffer to beads
b. Room temp shaker 10min
c. Spin down beads 1000rpm 4min
d. Retain supernatant (eluate) in new tube
e. Sample: eluate 10µL
f. Repeat for total of 3 elutions
g. Sample: 6uL eluted beads + 6uL H2O
h. Save beads at 4˚ for regeneration

12. Protease cleavage of GST fusion protein: Add 10X cleavage buffer to 1X final concentration + DTT to 1mM + 25µL (aliquot) of Prescission Protease. Rotator 4˚ overnight. Take a 10µL sample after cleavage.

13. Gel of results: 10% SDS-PAGE
a. Add dye to each sample (3µL of 5X dye or 10µL of 2X)
b. Heat 15 min at 100˚ (don’t let them boil dry!)
c. Run gel 200-250V 40-50 min (until dye is just leaving gel)
d. Stain with Coomassie
e. If protein is soluble and was cleaved well from the GST, move on to S-column purification!

14. S-column purification
a. Filter cleaved protein with 0.45µm, then 0.2µm syringe filter.
b. Transfer to Superloop, bring up to 50mL with S buffer A
c. Run on Äkta, mono S column (Andy: monoS32 p14 change to 1ml from 1.5ml)
d. Collect flow-through during loading and column rinse
e. Run 10% SDS-PAGE gel of load, flow-through, peak fractions
i. Load/ft samples: 10µL + 2µL 5X dye
ii. Fraction samples: 6µL + 6µL 2X dye

15. Concentration
a. Pool best fractions (cleanest/most concentrated). Dilute 1:1 with S buffer A (1mL buffer for each 1mL fraction). [I do this to decrease the salt concentration of the fractions—h65 hates high salt when the protein concentration is high, and it elutes from the S column at ~700mM NaCl.]
b. Concentrate in Centricon YM-30 until you can't stand it any more.
c. Measure protein concentration by Bradford / Bio-Rad assay

16. Dialysis: overnight at 4˚C (note: h65 precipitates in lower salt!). Measure protein concentration before distributing into aliquots. Freeze aliquots in N2(l), and store at –80˚.

Personal tools