Knight:Purification of His-tagged proteins/Denaturing

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(Procedure)
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#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
-
#*Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day.  From looking at the culture, it was near but not at saturation but I didn't have time to take OD readings.
+
#*''Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day.  From looking at the culture, it was near but not at saturation but I didn't have time to take OD readings.''
#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins.
#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins.
-
#*The Qiagen protocol didn't specify a temperature so I did 4°C.
+
#*''The Qiagen protocol didn't specify a temperature so I did 4°C.''
#Decant supernatant.
#Decant supernatant.
#The cell pellet can be stored at -70°C or processed immediately.
#The cell pellet can be stored at -70°C or processed immediately.
-
#*I stored the pellet at -80°C.
+
#*''I stored the pellet at -80°C.''
-
#Thaw for 15 mins.
+
#Thaw for 30 mins.
-
#*I thawed at room temperature since the next step happens at room temperature.
+
#*''I thawed at room temperature since the next step happens at room temperature.''
 +
#*''The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.''
 +
#Transferred to 2mL eppendorf tube.
#Resuspend in 1mL Lysis Buffer (see above).
#Resuspend in 1mL Lysis Buffer (see above).
-
#*Transferred to 2mL eppendorf tube.
 
#Incubate cells with agitation for 1 hr at room temperature.
#Incubate cells with agitation for 1 hr at room temperature.
-
#*Used an orbis shaker on the bench to do this temp (usually kept in 37° incubator).
+
#*''Used an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.''
==Notes==
==Notes==

Revision as of 11:41, 16 July 2006

In progress! If you want to purify His-tagged proteins, I recommend using Sauer:Purification of His-tagged proteins not this protocol!

This page is really just for notes purposes for Reshma.

Contents

Overview

"The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to both the specificity of the interaction between histidine residues and immobilized nickel ions and to the strength with which these ions are held to the NTA resin." [1]

Materials

Lysis and column equilibration buffer (Qiagen buffer B)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 8.0

Wash buffer (Qiagen buffer C)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 6.3

Elution buffer (Qiagen buffer E)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 4.5

Notes

  • Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H 2O you start with (maybe 50% of final volume)
  • When initially made up the solution had a pH of 5.88.
  • The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
  • The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.

Procedure

  1. Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
  2. The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
    • Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day. From looking at the culture, it was near but not at saturation but I didn't have time to take OD readings.
  3. Harvest the cells by centrifugation at 4000 x g for 15 mins.
    • The Qiagen protocol didn't specify a temperature so I did 4°C.
  4. Decant supernatant.
  5. The cell pellet can be stored at -70°C or processed immediately.
    • I stored the pellet at -80°C.
  6. Thaw for 30 mins.
    • I thawed at room temperature since the next step happens at room temperature.
    • The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.
  7. Transferred to 2mL eppendorf tube.
  8. Resuspend in 1mL Lysis Buffer (see above).
  9. Incubate cells with agitation for 1 hr at room temperature.
    • Used an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.

Notes

  • Sauer lab uses a Qiagen Ni NTA resin but we have an old kit with spin columns.

Safety

  • Some buffers may have guanidine hydrochloride in it.

References

  1. Qiagen Ni NTA Spin Kit manual [QiagenNTAManual]
  2. Sauer:Purification of His-tagged proteins/Denaturing prep [SauerDenaturingProtocol]
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