Daniel:Notebook/ComboLock/2016-5-26

From ZhangLabWiki
Revision as of 20:17, 25 May 2016 by >Djacobse (Created page with "=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 o...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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. Antibody Activation
    1. Resuspend VIM with 100 uL PBS (final conc 1 mg/mL)
    2. Equilibrate a 40 kDa spin column 4 times with 200 uL PBS, (100 mM pH 7.3)
      1. Spin at 1000xg for 2 min
    3. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
    4. Add 2 uL sulfo-SMCC to sample
    5. Incubate for 2 hr at 4C with 3 times intermittent mixing
    6. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
    7. Transfer antibodies to new column and spin for 3 min at 1000xg
  2. Oligonucleotide Activation