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

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
Line 35: Line 35:
<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 [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS</li>
<li>Remove excess DTT  using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS (10X PBS)</li>
<ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol>
<ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol>
</ol>
</ol>

Revision as of 22:23, 26 May 2016

VIM Antibody Prep

Back to Calendar

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. Prep
    1. Resuspend VIM-PA in 588 uL nfH2O
    2. Resuspend VIM-PB in 532 uL nfH2O
  2. 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 100 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
  3. 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 Zeba 7 kDa columns equilibrated with 100 mM PBS (10X PBS)
        1. Spin at 1500xg for 1 minute for equilibrium; 2 min for sample
  4. Ab-Oligo Mixing
    1. For VIM-PA and VIM-PB:
      1. Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
      2. Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
      3. Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
      4. Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl