Team:Washington/Notebook/IMAC protocol
From 2009.igem.org
(Difference between revisions)
Line 33: | Line 33: | ||
***Add 200uL of elution buffer pipette up and down to mix the beads | ***Add 200uL of elution buffer pipette up and down to mix the beads | ||
***Incubate for 2min the spin at 500rpm for 1min | ***Incubate for 2min the spin at 500rpm for 1min | ||
- | ***'''THE FLOW THROUGH IS | + | ***'''THE FLOW THROUGH IS YOUR PURIFIED PROTEIN!''' |
+ | *Recover the beads for re-use (~45min) | ||
+ | **Extract beads using 200uL diH2O and transfer to 15mL falcon tube | ||
+ | ***Store columns in dry place for later use after thoroughly washed out with water. It is fine to just run tap water over the columns after beads have been removed | ||
+ | **Spin down 800rpm for 5min, then decant supernatant | ||
+ | **Fill falcon tube with 100mM EDTA, and re-suspend beads | ||
+ | **Spin down 800rpm for 5min, then decant supernatant | ||
+ | **Fill falcon tube with diH2O, and re-suspend beads | ||
+ | **Spin down 800rpm for 5min, then decant supernatant | ||
+ | **Fill falcon tube with 100mM NiSO4, and re-suspend beads | ||
+ | **Spin down 800rpm for 5min, then decant supernatant | ||
+ | **Fill falcon tube with diH2O, and re-suspend beads | ||
+ | **Spin down 800rpm for 5min, then decant supernatant | ||
+ | **Add 20% ethanol until a 50% slurry has been reached. | ||
+ | **Store at 4C | ||
+ | |||
+ | ---- | ||
+ | |||
+ | *Buffer Examples (Alter to fit your protein's ideal buffer) | ||
+ | **Wash Buffer | ||
+ | ***1x PBS | ||
+ | ***30mM Imidizole | ||
+ | **BUGBUSTER Lysis Buffer | ||
+ | ***1x PBS | ||
+ | ***1mM PMSF | ||
+ | ***2x Bug Buster | ||
+ | ***2mg/mL lysozyme | ||
+ | ***0.2mg/ml DNAse | ||
+ | **Elution Buffer | ||
+ | ***1x PBS | ||
+ | ***250mM Imidizole |
Revision as of 00:45, 17 October 2009
BioRad Micro Column Protein Prep Protocol
- Day 1: Overnights
- Pick a single colony from plate and inoculate 1mL fresh media, with proper antibiotic, in 15mL culture tube
- Place lid on tube as not to form an air tight seal
- Shake at 37C for 16-24 hours at 200+ rpm
- Day 2: Expression
- Inoculate 50mL media with 500uL over night, in 250mL flask
- Shake 250mL flask at 37C at 200+ rpm
- Monitor OD600 of culture until OD600 ~0.4
- Use 200uL of culture and 800uL water in 1mL cuvette
- Read OD600 and multiply by 5 t get original OD600
- Should take ~2-3 hours
- INDUCE culture once culture gets to OD600 ~0.4 with IPTG so that the final concentration of IPTG is 0.5mM (ie 25uL 1M IPG for 50mL culture)
- Place in shaker 24-36hr at 18C.
- Day 3: STORE
- Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
- Store cells in -20C until ready for purification
- Day 3/4: Purification
- Lysis (~1hr)
- Add 1mL of lysis buffer, re-suspend, transfer to a 2mL eppindorf tube
- Continue to incubate at room temperature for 20min, mixing with plate mixer
- If worried about protein stability then incubate in cold room for 1hr on rocker
- Spin the lysis for 30-60min at 15000rpm (spin longer if supernatant is not clear, but 1hr should be plenty!)
- Transfer supernatant to a fresh tube (~1800uL)
- Purification (~1hr)
- Add 100uL of NiNTA Agarose 50% slurry to each column (this is the Ni-beads)
- Add 600uL of diH2O to each column
- Spin 500rpm for 10s, discard flow through
- Add 500uL of wash buffer, spin, discard flow through
- Add 500uL of supernatant, spin, discard flow through
- Repeat previous step until all supernatant has passed over column (~4x)
- CHANGE TO FRESH COLLECTION TUBE
- Add 200uL of elution buffer pipette up and down to mix the beads
- Incubate for 2min the spin at 500rpm for 1min
- THE FLOW THROUGH IS YOUR PURIFIED PROTEIN!
- Lysis (~1hr)
- Recover the beads for re-use (~45min)
- Extract beads using 200uL diH2O and transfer to 15mL falcon tube
- Store columns in dry place for later use after thoroughly washed out with water. It is fine to just run tap water over the columns after beads have been removed
- Spin down 800rpm for 5min, then decant supernatant
- Fill falcon tube with 100mM EDTA, and re-suspend beads
- Spin down 800rpm for 5min, then decant supernatant
- Fill falcon tube with diH2O, and re-suspend beads
- Spin down 800rpm for 5min, then decant supernatant
- Fill falcon tube with 100mM NiSO4, and re-suspend beads
- Spin down 800rpm for 5min, then decant supernatant
- Fill falcon tube with diH2O, and re-suspend beads
- Spin down 800rpm for 5min, then decant supernatant
- Add 20% ethanol until a 50% slurry has been reached.
- Store at 4C
- Extract beads using 200uL diH2O and transfer to 15mL falcon tube
- Buffer Examples (Alter to fit your protein's ideal buffer)
- Wash Buffer
- 1x PBS
- 30mM Imidizole
- BUGBUSTER Lysis Buffer
- 1x PBS
- 1mM PMSF
- 2x Bug Buster
- 2mg/mL lysozyme
- 0.2mg/ml DNAse
- Elution Buffer
- 1x PBS
- 250mM Imidizole
- Wash Buffer