We produced mAbs at my lab (classical 1975 hybridoma tech). One of my clones produced one mAb that was promising. When we constructed it, we used subtractive immunization, which consists of immunizing the mice with a non-tumoral cell line, then giving them a chemotherapy agent following 24h and 48h in order to eliminate proliferating B cells (triggered by exposition to non-tumoral cells). Then, we perform, at the same mouse, a series of immunizations with tumoral cells (same tissue-line cell). After purification we did the SDS page gel with the purified mAb as the sample. Strong IgG bands were easily spotted (150kda, 55kda and 22 kda). This mAb, when used in blotting, diluted at 1:1000 gave us the attached figure. First lane is a non-tumoral cell lysate (RWPE-1), second lane is the tumoral cell lysate PC3 (used as immunogen to produce the mAb) and third lane is another tomoral cell lysate, LNCAP. Here is how I am interpreting it: i) at the first lane, as expected, there is only low non-specific binding. At the other lanes, we have a more strong band (which I suppose is the protein my mAb recognize) but also a lot of other bands. I was expecting to see only one band, because my mAb is trully monoclonal. What am I missing here? Am I interpreting this correctly? Are these bands only a result of poor standardization of my blott? Should I do another blott, with both the primary and secondary antibodies more diluted? Thanks a lot!
In my opinion this antobody fraction is a) not containing only the one specific tpe of mAbs or b) your mAbs are in general not specific for your target protein
The bands in all three lanes are the same, the intensity is different. In my opinion your mAbs doesn’t bind specifically to its target. I also recommend checking the lysates! Your target protein can be degraded somehow.
Good Luck
But the lanes come from different cell line lysates....how can they be all the same?
sometimes the obtained band pattern look somewhat different (compare lane 1 vs 3, in the upper middle section). However, in my opinion your antibodies do not work. The mAbs detect various proteins which may contain your target sequence (at least partially).
Could this be direct binding of the secondary antibody to the bands? I was going to test another secondary antibody just in case....cause I find it interesting that there is clearly a band that is much stronger (right at the middle of the lanes) then the others. I thank you all for your considerations so far. I find this a very good place to discuss and learn, because even if we get judged it doesn't really matter since we are so far away from each other. =)
Andrei It sure could be ,try running only the secondary antibody as a control. It is always best if you used pre-absorbed antibodies. That should eliminate your issue.
Good Luck, Butch
Andrei Moroz I think your idea about the secondary Ab is probably the most likely: do another blot with an unrelated mAb AND one with NO primary Ab, and you'll see your answer. Or absorb the secondary with the non-tumoral cell lysate before use (see protocol here: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=2ahUKEwiboMPRrN7gAhWGVhUIHWFwA68QFjAAegQICBAC&url=https%3A%2F%2Fopen.uct.ac.za%2Fbitstream%2Fhandle%2F11427%2F25146%2FMolecular%2520Methods.pdf%3Fsequence%3D1%26isAllowed%3Dy&usg=AOvVaw0KJ__J9Lil2nzixZ4qTkV_)
Thank you all for ur kind suggestions. I will get to work and later on will post the new blotting here.
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