MAKING AN ANTIBODY

Beth 3/02

1) Figure out the best region of the protein that is exposed and more antigenic (programs like DNA star have predictions for these things). An epitope is 6 amino acids long.

2) Express this protein in BL21 cells of the like until you get enough for injection (call covance- usually it is a few mLs of 1.5mg/ml of protein, you can also submit it in gel form.

3) Once you are ready to start a rabbit or chicken, get a few pre-bleeds from the organism. Incubate these with blots that have lysate of your test organism (ie yeast or amoeba) and also have e. coli extract to make sure that nothing reacts in the same size as your immunogen. Then you can tell Covance which animal you want to use for your antibody project. Do not take long to do any of these animal steps since they charge for maintenance of the animals that you are testing….while they hold them until they hear back from you.

4) Send the antibody, enough for all their boosts (they will in return send you a schedule).

5) In the meantime, run out as many blots that you can stand, of E. coli lysate and lysate from the organism that you will be using the antibody on. Use the 15 well gels and run one ladder and the rest with prebleed, e.coli lysate or the other organism lysate. Once you Coomassie stain the blot, cut the blot into strips (15 of them) and dry and save the ladder lane in a particular notebook that you can store all the respective ladders. Make sure that you label each set of strips and ladder so that you know which ones go with each ladder. I usually use a different colored pencil and I make a line at the bottom of the blot prior to cutting it so that the strips and ladder can be lined up. I store the strips in milk (5% Milk in TBST plus azide) in the clear plates that are used for LB plates. Parafilm them, date them, code them- refer to where the ladder is and store them in the fridge. These strips keep indefinitely, if the milk starts to fall out of solution, just rinse the plate and strips in diH2O and replace the milk.

6) Once you get your test bleeds back from Convance or whereever, you now have a bunch or strips to monitor your progress against. I usually pick a few dilutions of the bleed to use on the different strips. I incubate the strips in those small culture tubes using milk as the diluent. Process for ECL as normal. Match up the strips with the ladder to be certain you have the correct size protein reacting. You will probably have to affinity purify the antibody.