Troubleshooting Protocol 062912

2015-01-13

azim58 - 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
Troubleshooting Protocol 062912.pdf

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

azim58wiki: