Daniel:Notebook/ComboLock/2016-5-26: Difference between revisions
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>Djacobse |
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<li>Do this twice:</li> | <li>Do this twice:</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>Equilibrate a 40 kDa spin column 4 times with | <li>Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3) | ||
<ol type="i"><li>Spin at 1000xg for 2 min</li></ol> | <ol type="i"><li>Spin at 1000xg for 2 min</li></ol> | ||
<li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube</li> | <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube</li> | ||
Line 31: | Line 31: | ||
<li>For VIM-PA and VIM-PB:</li> | <li>For VIM-PA and VIM-PB:</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>Add | <li>Add 13 uL 100 uM oligonucleotide to tube</li> | ||
<li>Add | <li>Add 22 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 | <li>Add 165 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 [[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> | </ol> | ||
</ol> | </ol> | ||
Line 41: | Line 42: | ||
<ol type="A"><li>For VIM-PA and VIM-PB:</li> | <ol type="A"><li>For VIM-PA and VIM-PB:</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides | <li>Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)</li> | ||
<li>Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup</li> | <ol type="i"><li>Amounts to 10X molar excess of oligo to Ab</li> | ||
<li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li> | |||
<li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li> | <li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li> | ||
<li>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</li> | <li>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</li> | ||
</ol> | </ol> |
Latest revision as of 22:38, 26 May 2016
VIM Antibody Prep[edit]
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[edit]
- Prep
- Resuspend VIM-PA in 588 uL nfH2O
- Resuspend VIM-PB in 532 uL nfH2O
- 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 100 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 13 uL 100 uM oligonucleotide to tube
- Add 22 uL 40 mM DTT
- Incubate at 95C for 2 min followed by 1 hr at 37C
- Add 165 uL PBS with 20 mM EDTA
- Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS (10X PBS)
- Spin at 1500xg for 1 minute for equilibrium; 2 min for sample
- Ab-Oligo Mixing
- For VIM-PA and VIM-PB:
- Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)
- Amounts to 10X molar excess of oligo to Ab
- Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
- Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
- 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