hello to everybody.
i'm planning to incubate the membrane overnight with 3 different antibodies made in the same specie.
the three protein are different in size .. so I don't understand why it shouldn't work
does someone experience with this ?
thank you Gianluca
You can, but you must perform the apropiate parallel controls.
One of you samples should by loaded 4 extratimes that will be cutted. One for each antibody and the fourth for the negative control.
Mandatory: as a negative control use unspecific IgG of the same origin as your primary antibody at the same concentration as your primary antibodies. This IgGs are not expensive and very valuable.
If you use a cocktail of primaries sume up the concentrations to obtain the IgG that should go to the negative control. (If the primaries are of differen origin then you must mix IgG from all those origins).
You may-will find yourself shocked with the bands that you supposed good but are unspecific.
This unspecificity come from the primary itself, from the simple fact that it is an antibody no matter which epitope or purification.
The reason is antibodies (IgGs) have disulfur bonds and charged aminoacids.
To avoĦd this extra-bands you can tryto preincubate your antibody with iodoacetic acid or reduced glutatione. this will quench free sulphiydryls. To avoid charges interaction you can ad tween or more salt.
Of course sometimes the antibody contain non-antibody binding stuff (other antibodies or proteins). With primaries there isn't much to do about it but with secondary I'd advice to buy only affinity purified and Fab fragment if possible.