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.