Western AB unselective or posttranslational modifications? - (May/18/2006 )
Hi,
I've been doing Westerns for two months now with a polyclonal anti-cytoglobin or anti-neuroglobin antibody respectively.
As we always get multiple bands besides from the expected height, we used to say: ok, antibody is too unselective.
Couldn't it also be posttranslational modification of protein as bands run higher than cygb/ngb and don't have the right weight for multimers?
How can I test that? Do I have to do native page, cut bands and sequence???
Never done that before and we don't have the equipment for automated edman so I'd be glad if there was a simpler alternative.... 
hello,
the bands you see on your western could result from cross-reactivity against non-specific proteins by either a. ) your primary antibodies, or b.) your secondary Abs. to rule out "b", try probing your membrane with secondary Ab alone as a negative control. if, on the other hand, your extra bands result from the type of primary Ab that you're using, then you might want to use a negative control (pre-immune serum would be the best choice). also, are your primary Abs affinity purified? if not, you could just be seeing background bands, and not real post-translational modifications (PTMs).
note: ammonium sulfate, and protein a/g might not get rid of this type of cross-reactivity. immunoadsorption against purified protein will be the best route to clean Abs.
still, if your Abs are good, then you may be dealing w/ PTMs. if you are, you should use your Abs for a large scale immunoprecipitation experiment. you could then test your eluate using a series of stains (for phophorylation, glycosylation, etc). alternatively, you could slice the bands out of your stained gel (if you get enough protein in your eluate) and have them sequenced by mass spec. a 1D or 2D gel will work fine for this. no need for native gels. also, don't try this w/ edman sequencing.
i see you're from germany, and so i'm pretty confident that there are some good mass spec labs at max plank that you could contact about this if you think you need to identify the types of modifications you're seeing. if you can pull down a lot of protein with your Abs, see if they can analyze your proteins by both MALDI-TOF and LC-MS/MS. this will give you more coverage of your proteins than by using mass spec analysis from only one type of ionization source.
still, i would first to a thorough check of your antibodies before making the conclusion that you have PTMs.
i hope this helps,
jon
I've been doing Westerns for two months now with a polyclonal anti-cytoglobin or anti-neuroglobin antibody respectively.
As we always get multiple bands besides from the expected height, we used to say: ok, antibody is too unselective.
Couldn't it also be posttranslational modification of protein as bands run higher than cygb/ngb and don't have the right weight for multimers?
How can I test that? Do I have to do native page, cut bands and sequence???
Never done that before and we don't have the equipment for automated edman so I'd be glad if there was a simpler alternative....