Basic Story 2-6-12
2014-08-29azim58 - Basic Story 2-6-12
2-6-12 As of this date, we have tried purifying antibodies using a random
peptide using TentaGel beads, but the resulting purified antibody could
not bind our targets well. We then decided we would try to purify the
antibody first. We tried purifying with a protein A column, but this type
of column does not purify all sub-types of the IgG as well. Bart ordered
a protein G column which generally purifies all of the IgG antibodies
better. We will try to purify with that, and then we will see how well
the purified antibody binds to the random peptide when it is on a glass
slide in a chamber with a pump.
2-8-12 Attempted to purify antibodies with Protein G column. We didn't
get as much antibody as we would like. We will get more sera from mice
and repeat the experiment to get more antibody. On the next attempt, we
will most likely incubate the antibody with the protein G longer as well.
See IgG Purification with Protein G
2-20-12 Bart quantitated the amount of antibody reacting with the TOR1
random peptide. In Y mL sera (maybe about 1 mL) there was only about 192
ug antibody. In order to get enough antibody Bart said that he would need
about 25-30 mL (he earlier obtained 25 mL from 88 mice). 192 ug/mL*30mL =
5760 ug. I'm not sure if I have all of these numbers right. Anyway, he
thinks if we can get enough sera (from about 100 naive mice), we might be
able to get enough antibody to purify on a column.
2-27-12 Bart continued to perform the dialysis of the TOR antibodies. Woa
tried to see if KA10 antibody was present with a Western blot, but they
didn't see any bands. Bart would like to test for binding of antibody
with a peptide on a slide with a flow cell. He is also investigating
whether he can buy naive sera from a company. He is also interested in
how we could quantitate how much antibody is binding to a peptide. He is
also considering using different types of beads such as some sepharose
beads mentioned in the current protocols in immunology protocol.