User:Matt Hartings/Notebook/Photosynthesis/2012/06/26: Difference between revisions

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==Objective==
==Objective==
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Purify Hemoglobin extracted and dialyzed [[User:Matt_Hartings/Notebook/Photosynthesis/2012/06/25|yesterday]] and extract and dialyze WT Hb that Tamra [[User:Tamra_L._Fisher/Notebook/Research_for_Dr._Hartings/2012/06/22|expressed]].


==Description==
==Description==
# Add experimental record here. Include what, how, and why...
 
==Purifying WT Hb==
The wt hemoglobin was extracted at very high concentrations (i.e. the extract was a deep red color). I had roughly 70 mL to purify. I ran 10 mL at a time through the column.
# Wash column with water to remove ethanol
# Equilibrate the column with binding buffer (25mM Tris, 50mM NaCl, pH 8.3) - Note: I had never used this pH before. But it was suggested in a [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0020176 paper] by Storz on the optimization of hemoglobin expression in E coli. Also, in an email note from Storz and his postdoc, I learned that they add hemin in DMSO. Should be worth checking out.
# Run 10mL of of protein sample on the column
# Run 5mL of binding buffer over the column
# Elute with at 0-50% gradient (elution buffer = 25mM Tris, 1M NaCl, pH 8.3) over 30 minutes. Note: the elution buffer was on pump A and the binding buffer was on pump B.
# Clean column with 100% elution buffer
# Re-equilibrate column with 100% binding buffer
# Repeat until all protein is purified
 
[[Image:20120626aFPLC WTAscHb.PNG|600px]]
 
[[Image:20120626bFPLC WTAscHb.PNG|600px]]
 
The two sets of FPLC runs were separated because I took a break for lunch and didn't want the UV detector running while I was gone. The desired protein fractions were pooled and stored at 4C.
 
After the final elution I cleaned the system.
# Wash with water to remove salts
# Wash with 20% ethanol to ensure nothing grows in system
# Add 20% ethanol to super-loop
 
==Extracting Tamra's Hemoglobin Expression==
 
Tamra had expressed hemoglobin, and, instead of using hemin in DMF, she used a hemin stock that I had made.
 
The hemin stock was prepared by:
# Measure 160mg of hemin
# Dissolve/incorporate the hemin into 2.75mL of triethanolamine.
# Add water to 50mL
# Adjust the pH to ~7.8 with HCl
## I used pH strips for this
## If you go below this pH, the hemin crashes out as the dichloride.
# Add water to 150mL
# Filter in a syringe filter
 
The hope with doing this was that the expression would work as well, and there wouldn't be lots of hemin and hemin byproducts left after centrifuging, which seem to be causing my Q columns to turn black.
 
Extraction
# Resuspended cells were thawed
# Cells were lysed with the ultrasonicator
## 30 seconds of sonicating at power level 12
## 30 seconds on ice
## Repeat for a total of three cycles
# Cells were added to centrifuge tubes
# Tubes were balanced to near 0.01g
# Tubes were centrifuged for 2 hours at 18000rpm and 4C
# Supernatant was dialyzed overnight into 25mM Tris, 50mM NaCl, pH 8.3
 
Note: the extract here was not as red as the extract that I had when adding hemin in DMF at induction.


==Data==
==Data==

Revision as of 06:34, 28 June 2012

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Objective

Purify Hemoglobin extracted and dialyzed yesterday and extract and dialyze WT Hb that Tamra expressed.

Description

Purifying WT Hb

The wt hemoglobin was extracted at very high concentrations (i.e. the extract was a deep red color). I had roughly 70 mL to purify. I ran 10 mL at a time through the column.

  1. Wash column with water to remove ethanol
  2. Equilibrate the column with binding buffer (25mM Tris, 50mM NaCl, pH 8.3) - Note: I had never used this pH before. But it was suggested in a paper by Storz on the optimization of hemoglobin expression in E coli. Also, in an email note from Storz and his postdoc, I learned that they add hemin in DMSO. Should be worth checking out.
  3. Run 10mL of of protein sample on the column
  4. Run 5mL of binding buffer over the column
  5. Elute with at 0-50% gradient (elution buffer = 25mM Tris, 1M NaCl, pH 8.3) over 30 minutes. Note: the elution buffer was on pump A and the binding buffer was on pump B.
  6. Clean column with 100% elution buffer
  7. Re-equilibrate column with 100% binding buffer
  8. Repeat until all protein is purified

The two sets of FPLC runs were separated because I took a break for lunch and didn't want the UV detector running while I was gone. The desired protein fractions were pooled and stored at 4C.

After the final elution I cleaned the system.

  1. Wash with water to remove salts
  2. Wash with 20% ethanol to ensure nothing grows in system
  3. Add 20% ethanol to super-loop

Extracting Tamra's Hemoglobin Expression

Tamra had expressed hemoglobin, and, instead of using hemin in DMF, she used a hemin stock that I had made.

The hemin stock was prepared by:

  1. Measure 160mg of hemin
  2. Dissolve/incorporate the hemin into 2.75mL of triethanolamine.
  3. Add water to 50mL
  4. Adjust the pH to ~7.8 with HCl
    1. I used pH strips for this
    2. If you go below this pH, the hemin crashes out as the dichloride.
  5. Add water to 150mL
  6. Filter in a syringe filter

The hope with doing this was that the expression would work as well, and there wouldn't be lots of hemin and hemin byproducts left after centrifuging, which seem to be causing my Q columns to turn black.

Extraction

  1. Resuspended cells were thawed
  2. Cells were lysed with the ultrasonicator
    1. 30 seconds of sonicating at power level 12
    2. 30 seconds on ice
    3. Repeat for a total of three cycles
  3. Cells were added to centrifuge tubes
  4. Tubes were balanced to near 0.01g
  5. Tubes were centrifuged for 2 hours at 18000rpm and 4C
  6. Supernatant was dialyzed overnight into 25mM Tris, 50mM NaCl, pH 8.3

Note: the extract here was not as red as the extract that I had when adding hemin in DMF at induction.

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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