Troubleshooting+Protocol+062912

6-29-12

It looks like there are many possible modifications that could be tried.
 * try using tentagel beads with 4X the peptide density. This can be accomplished by putting 2 lysines at the beginning of the peptide sequence and blocking 2 times with Fmoc rather than Fmoc and Boc as explained by Zhao.
 * use more sera (I used 10 uL in 1000 uL equilibration buffer last time). I could try using closer to 1 whole mL
 * try doing the whole procedure in a 1.5 mL tube as recommended by Shen rather than a 5 mL column. If I do use a column, I should use a smaller column than a 5 mL column.
 * try incubating the sera with the beads at 4 C overnight rather than at 1 hr at 37 C or try both.
 * try Shen's Thermo binding and elution buffers rather than the handmade buffers
 * Clear the sera before using by centrifuging? I think Shen may have done something like this.

Here are some sketches from Shen and Zhao

e-mail to Kathy about troubleshooting: https://mail.google.com/mail/u/0/?ui=2&shva=1#sent/1384385758a17c65