User:Matt Hartings/Notebook/Photosynthesis/2012/06/30: Difference between revisions
From OpenWetWare
(Autocreate 2012/06/30 Entry for User:Matt_Hartings/Notebook/Photosynthesis) |
No edit summary |
||
Line 7: | Line 7: | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Objective== | |||
Purify crude sample from AA001MRH synthesis from [[User:Matt_Hartings/Notebook/Photosynthesis/2012/05/16|May 16]] (might have waited too long on this) and extract WT hemoglobin from yesterday's expression and [[User:Tamra_L._Fisher/Notebook/Research_for_Dr._Hartings/2012/06/29|continuation]] of Tamra's Hb cobalt-protoporphyrinIX reconstitution. | |||
==Procedure== | |||
Purification in overall synthesis of L-azidohomoalanine (full protocol found [http://www.nature.com/nprot/journal/v5/n12/abs/nprot.2010.164.html here] | |||
# Prep column | |||
## Place glass wool in methanol to soak and place in the bottom of the 8cm diameter column | |||
## Top the glass wool with ~1cm sand | |||
## Prep the column material | |||
### 200mL of silica (Note: next time use more ... perhaps 300mL) | |||
### Prepare mobile phase 500mL of 99:1 Dichloromethane:Methanol (Overall I used around 1-1.5L for the column) | |||
### Add enough of the mobile phase to the silica to make a pourable slurry | |||
## Pour the silica slurry into the column | |||
## Let settle for a few minutes | |||
## Pressurize the column with air to pack the column and run out some of the mobile phase. | |||
# Run the mobile phase through the column until there is just a little liquid remaining on top of the column. | |||
# Dissolve the product (oil) from [[User:Matt_Hartings/Notebook/Photosynthesis/2012/05/16|Reaction AA001MRH]] in the mobile phase. | |||
# Add the reaction product to the column with a pasteur pipette. | |||
# Run the product into the column until there is no more liquid left on the top of the column. (Don't let it dry) | |||
# Add ~ 2cm of sand to the top of the column. | |||
# Add more mobile phase to the top of the column | |||
# Run the mobile phase through the column and collect fractions. (I collected 22 fractions) | |||
# Run TLC on the fractions | |||
# A compound appeared in fractions 6-15 (with the same RF) (this volume amounted to roughly 250mL) | |||
## Note: the Nature protocol paper from above mentions that the product should have a RF of around 11%. My fractions appeared to have 2 different chemicals in solution with RFs of ~20% and 50%. | |||
# Rotovap fractions | |||
# Rotovapping resulted in an oil (as suggested by the Nature Protocol paper) of a similar volume to the oil that I began the purification with (judging by eye). | |||
# The oil (in a 500mL round bottom flask) was stoppered and placed in the fridge. | |||
Extraction of Hemoglobin | |||
# Thaw cells | |||
# Sonicate on power level 12 | |||
## 30 seconds on | |||
## 30 seconds on ice | |||
## repeat twice | |||
# Add cells to centrifuge tubes and balance to ~0.01g | |||
# Centrifuge for 2 hours at 18000rpm and 4C | |||
# Prepare dialysis buffer (25mM Tris, 50mM NaCl, pH 8.3) | |||
## 12.114 g Tris | |||
## 11.688 g NaCL | |||
## 4L water | |||
## Adjust pH with 3M HCl | |||
# Place Hb in dialysis bags and add to dialysis buffer | |||
# Leave in cold room overnight | |||
Continuation of Hb Co-protoporphyrinIX reconstitution | |||
# Tamra had already made the Co-protoporphyrinIX solution to be added. | |||
# I added this solution dropwise to the hemoglobin solution in the cold room and covered with aluminum foil | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 12:45, 30 June 2012
Biomaterials Design Lab | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectivePurify crude sample from AA001MRH synthesis from May 16 (might have waited too long on this) and extract WT hemoglobin from yesterday's expression and continuation of Tamra's Hb cobalt-protoporphyrinIX reconstitution. ProcedurePurification in overall synthesis of L-azidohomoalanine (full protocol found here
Extraction of Hemoglobin
Continuation of Hb Co-protoporphyrinIX reconstitution
|