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&shva=1#sent/1384385758a17c65