Any experieces with unstable primary antibodies? - still recognizes recombinant protein but not native... HELP (Jul/11/2006 )

Has anybody ever experienced this kind of problem:

primary antibody (polyclonal, self-isolated from serum, will look up elution buffer....)
tested and worked well somehow loses the ability to recognize the antigen in samples
signal for recombinant protein still comes up though a bit weaker than directly after purification and there are still unspecific bands in the lanes of samples but none on the height of recombinant protein

we thought ab was quickly degrading when thawed so we now directly add sodium azide but no change...

can't be the samples cause same sample (already in loading buffer) sometimes gives good signals and sometimes not

I'll attach some pics as soon as I'm in the lab...

at the moment I'm incubating with these antibodies for 3 times which means once over night (now room temperature instead of 4°C) and develop film: very weak signal (only recombinant and unspecific bands), so back into incubation with fresh antibody again over night rt: signals stronger but still no specific band apart from recombinant protein, so now on 2 days incubation rt...

primary ab dilution is 1:500 and I'm slowly running out of antibody

so attached are the pics:

first one with strong recombinant signal and signals in the other lanes (same sample in different concentrations loaded)

second very weak rekombinant signal and no sample signals at all (different samples, same tissue)


antibody affinity isolated: elution with 1ml 100mM Glycin pH 2,5 with reaction tube already containing 100 mikrol 1M Tris Hcl