Daniel:Notebook/ComboLock/2016-5-26: Difference between revisions
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<li>For VIM-PA and VIM-PB:</li> | <li>For VIM-PA and VIM-PB:</li> | ||
<li>Add 1.3 uL | <ol type="a"> | ||
<li>Add 1.3 uL 100 uM oligonucleotide to tube</li> | |||
<li>Add 2.2 uL 40 mM DTT</li> | <li>Add 2.2 uL 40 mM DTT</li> | ||
<li>Incubate at 95C for 2 min followed by 1 hr at 37C</li> | <li>Incubate at 95C for 2 min followed by 1 hr at 37C</li> | ||
<li>Add 20 uL PBS with 20 mM EDTA</li> | <li>Add 20 uL PBS with 20 mM EDTA</li> | ||
<li>Remove excess DTT using two consecutive 7 kDa columns equilibrated with 100 mM PBS</li> | <li>Remove excess DTT using two consecutive 7 kDa columns equilibrated with 100 mM PBS</li> | ||
</ol> | |||
</ol> | </ol> | ||
</ol> | </ol> |
Revision as of 20:28, 25 May 2016
VIM Antibody Prep
I found some columns Dinh had that were the product I've been waiting on, so I am going to use those to test out the antibody linkage and binding protocols. I'll use the VIM protein again, since it has the highest expression.
Protocol
- Antibody Activation
- Resuspend VIM with 100 uL PBS (final conc 1 mg/mL)
- Do this twice:
- Equilibrate a 40 kDa spin column 4 times with 200 uL PBS, (100 mM pH 7.3)
- Spin at 1000xg for 2 min
- Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
- Add 2 uL sulfo-SMCC to sample
- Incubate for 2 hr at 4C with 3 times intermittent mixing
- Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
- Transfer antibodies to new column and spin for 3 min at 1000xg
- Oligonucleotide Activation
- For VIM-PA and VIM-PB:
- Add 1.3 uL 100 uM oligonucleotide to tube
- Add 2.2 uL 40 mM DTT
- Incubate at 95C for 2 min followed by 1 hr at 37C
- Add 20 uL PBS with 20 mM EDTA
- Remove excess DTT using two consecutive 7 kDa columns equilibrated with 100 mM PBS