Daniel:Notebook/ComboLock/2016-5-26: Difference between revisions

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>Djacobse
>Djacobse
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<ol type="A">
<ol type="A">
<li>For VIM-PA and VIM-PB:</li>
<li>For VIM-PA and VIM-PB:</li>
<li>Add 1.3 uL VIM-PA to tube</li>
<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

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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

  1. Antibody Activation
    1. Resuspend VIM with 100 uL PBS (final conc 1 mg/mL)
    2. Do this twice:
      1. Equilibrate a 40 kDa spin column 4 times with 200 uL PBS, (100 mM pH 7.3)
        1. Spin at 1000xg for 2 min
      2. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
      3. Add 2 uL sulfo-SMCC to sample
      4. Incubate for 2 hr at 4C with 3 times intermittent mixing
      5. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
      6. Transfer antibodies to new column and spin for 3 min at 1000xg
  2. Oligonucleotide Activation
    1. For VIM-PA and VIM-PB:
      1. Add 1.3 uL 100 uM oligonucleotide to tube
      2. Add 2.2 uL 40 mM DTT
      3. Incubate at 95C for 2 min followed by 1 hr at 37C
      4. Add 20 uL PBS with 20 mM EDTA
      5. Remove excess DTT using two consecutive 7 kDa columns equilibrated with 100 mM PBS