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I have some questions about this antibody. How much GFP could be combined to the cross-linked IgG for 20µl of slurry per IP? What is the condition to elute combined protein off beads ? I also want to know the how strong the conjugation is between antibody and sepharose? When protein is eluted, will the GFP antibody also be eluted off the beads?
Asked on May 27 2011
Replies to your questions: 1. 10ul Beads (20ul slurry) will contain 1ug IgG, each IgG (MW:150,000) can bind 2 GFP molecules (~MW:25,000 each ), therefore about 666ng of GFP should bind to these beads. Cross-linking should not interfere too much with the binding. 2. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). 3. The cross-link was carried out by a proprietary process that should minimize leaking of both IgG heavy and light chains. Typically less than 5% of the IgG content on the beads will leak. 4. By boiling samples there is a risk of having the antibody eluted too, therefore a mild eluent, such as glycine buffer, should be used to avoid this from happening.
Answered on May 31 2011