How to test new homemade antibody?
Posted 15 September 2010 - 05:06 AM
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?
Posted 15 September 2010 - 05:43 AM
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...
Posted 15 September 2010 - 11:42 PM
Posted 16 September 2010 - 01:17 AM
"This is SPARTA!"
"I´m the goddamn batman"