Technique To Affinity Purify Antibodies From Immunized Animal Sera Part 1

Hello,

My name is LeBlond, Gerard LeBlond and my purpose is to serve up detailed techniques that can be used to perform specific tasks in the Molecular Biology lab.

Recently I was commissioned with the task of raising antibodies against a new protein discovered in our lab.

Whose lab made this request? The research group is headed by David R. Mitchell, Ph.D. (principal investigator) of the Department of Cell & Developmental Biology at SUNY UMU in Syracuse, NY.

In this 4 part series of posts I shall reveal the protocol that I used to accomplish that task.

But first I should mention that the procedure, called: "Affinity Purification of Antibody From Sera" is meaningless by itself and in isolation from any other work.

For instance, in order to begin the project it is first necessary to isolate the previously uncharacterized protein (or you can start with a previously isolated protein and do the technique for practice... but what's the fun in that?), then you need to immunize lab animals (we used rabbits) using a specific protocol and you need to make certain you isolate blood serum prior to (this is, by convention, called the 'pre-bleed' sera though it could just as accurately be called the 'pre-immune' sera... but who am I to fight convention) exposing the animal to your protein (the antigen), and you need to take blood samples periodically after each antigenic boost.

You'll also need to size-separate your protein using SDS-polyacrylamide gel electrophoresis and adhere your protein onto an hydrophobic membrane using the Western Transfer procedure.

In subsequent posts I'll go over the policies and procedures associated with successfully performing each of those techniques but this series of missives is dedicated to affinity purification.

In Part 2 of the series of posts I will explain exactly what you need before you begin the procedure.

Part 3 will be dedicated to giving the sequential steps which I have followed to successfully affinity purify rabbit antibodies against my newly discovered protein.

And Part 4 will focus on providing helpful hints for making the process run efficiently and some of the tricky areas to pay particular attention.

So, from Central New York, this is LeBlond, Gerard LeBlond.


P.S. In order to respect the confidentiality and certain priveleged information between my associates I can not divulge the identity or specific characteristics of the newly discovered protein. That information can only see the light of day via the process of peer-reviewed publication in a mainstream scientific journal.

P.P.S. Of course, when the results are published I will be happy to quote the name of the publication and the dates and pages associated with the published work. But until then the word is "MUM".