How to test new homemade antibody? - (Sep/15/2010 )

So, it's time to go ahead with my little project... I'm supposed to get my newly homemade antibody in the next month... but I just figured out I have no clue how to really start testing it? Is there any general protocols, articles or anything, which would teach such a novice how to approach the project...

I think I could ran the purified protein (which was injected to the rabbit) on the gel and do normal Western blotting... then I have the protein (more or less) in the expression vector in the HEK cells, which means I could try to stain those cells... I also know more or less the localization of the protein in tissues and brain slices... but yet... I know many people have made their own antibodies, but how to do you test them are they even working?

When you try to test the specificity by Western blot you need a positive control and when possible a negative control.

Then you do two blots which contain both samples.
One blot you incubate with the antibody you like to show that it is specific.

For the second you preincubate your antibody with the purified protein it was raised against (you may do this over night and of course with a high concentration of protein). Then you do a centrifugation step to remove the precipitate (that you often do not see) and incubate the second blot with the supernatant.

The first blot should show a signal in the expected size of the positive control. In the negative control you should not see anything.
In the second blot the specific band of the posotive control should disappear, because you antibody was precipitated before. When you did not use enough protein, at least the signal should be much weaker than in the first blot.

For staining you may do the same thing...

I would start with ELISA and dilution "checkerboard" - ie. dilution series of both antibody and antigen. You do that by coating rows of wells with dilution series of antigen and then using dilution series of antibodies in columns. What you get from that is the series of dilution curves that give you some information on specificity of your antibodies.

What about peptide competition?