Sauer:Purification of His-tagged proteins/Denaturing prep: Difference between revisions
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This is a general protocol for purification of His<sub>6</sub>-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni<sup>2+</sup>-NTA resin from [http://www1.qiagen.com/SelectCountry.aspx Qiagen]. | This is a general protocol for purification of His<sub>6</sub>-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni<sup>2+</sup>-NTA resin from [http://www1.qiagen.com/SelectCountry.aspx Qiagen]. | ||
=Lysis= | |||
The amount of lysis buffer you use will depend on the number of cells you have. The following works well for cells harvested from a 1 L culture, and I never pay attention to the density of the culture. | |||
#Resuspend cell pellet in 10 mL lysis buffer. | |||
#Freeze at -80 ˚C for at least 30 min. | |||
#Thaw and bring lysis buffer volume to 40 mL. | |||
#Allow to lyse at room temperature for 30 min. | |||
#Spin to pellet the cellular debris at 14000 rpm for 30 min in a SA600 or similar rotor. | |||
#Retain the supernatent. | |||
=Purification= | |||
The amount of Qiagen Ni<sup>2+</sup>-NTA resin (or similar product) you use depends on the amount of protein you have in your culture. Less is typically better. The protocol below works for most proteins that were grown in a 1 L culture. | |||
#Add 1 mL of 50% Ni<sup>2+</sup>-NTA resin slurry to lysate. | |||
#Bind at room temperature, shaking gently, for 30 min–1 hr. | |||
#Spin at low speed to pellet the resin. | |||
#Remove supernatent and save (this is your flowthrough). | |||
#Add 7 mL of lysis buffer to the resin and transfer the resin to a small column. (I like these [http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@1901512925.1139437580@@@@&BV_EngineID=cccdaddgmgmmfiecfngcfkmdhkkdfll.0&categoryPath=%2fCatalogs%2fLife+Science+Research%2fChromatography+%7c+Protein+Purification%2fChromatography+Columns%2fEmpty+Columns%2fPoly-Prep+Columns&divName=Corporate&loggedIn=false&lang=English&country=HQ&catLevel=6&catOID=-31109&isPA=false&serviceLevel=Lit+Request&searchStr=731-1550&cateName=Ordering+Information columns] from Biorad.) | |||
#Collect wash. | |||
#Wash two times with 7 mL of wash buffer 1 (collect). | |||
#Wash with a total of 50 mL of wash buffer 2 (collect). | |||
#Elute four times with 1 mL of elution buffer (collect each separately). | |||
=Buffers= | =Buffers= | ||
===Lysis buffer=== | ===Lysis buffer=== |
Revision as of 15:30, 8 February 2006
Introduction
This is a general protocol for purification of His6-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni2+-NTA resin from Qiagen.
Lysis
The amount of lysis buffer you use will depend on the number of cells you have. The following works well for cells harvested from a 1 L culture, and I never pay attention to the density of the culture.
- Resuspend cell pellet in 10 mL lysis buffer.
- Freeze at -80 ˚C for at least 30 min.
- Thaw and bring lysis buffer volume to 40 mL.
- Allow to lyse at room temperature for 30 min.
- Spin to pellet the cellular debris at 14000 rpm for 30 min in a SA600 or similar rotor.
- Retain the supernatent.
Purification
The amount of Qiagen Ni2+-NTA resin (or similar product) you use depends on the amount of protein you have in your culture. Less is typically better. The protocol below works for most proteins that were grown in a 1 L culture.
- Add 1 mL of 50% Ni2+-NTA resin slurry to lysate.
- Bind at room temperature, shaking gently, for 30 min–1 hr.
- Spin at low speed to pellet the resin.
- Remove supernatent and save (this is your flowthrough).
- Add 7 mL of lysis buffer to the resin and transfer the resin to a small column. (I like these columns from Biorad.)
- Collect wash.
- Wash two times with 7 mL of wash buffer 1 (collect).
- Wash with a total of 50 mL of wash buffer 2 (collect).
- Elute four times with 1 mL of elution buffer (collect each separately).
Buffers
Lysis buffer
100 mM NaH2PO4
10 mM Tris base
6 M guanidine hydrochloride
10 mM imidazole
Adjust to pH 8
Wash buffer 1
100 mM NaH2PO4
150 mM NaCl
8 M urea
20 mM imidazole
Adjust to pH 8
Wash buffer 2
50 mM NaH2PO4
500 mM NaCl
20 mM imidazole
Adjust to pH 8
Elution buffer
50 mM NaH2PO4
500 mM NaCl
250 mM imidazole
Adjust to pH 8