Dear reader,

Maybe you can help us out w/ an IP-problem that we stumble upon.

We try to do an immuno precipitation of a protein (POI) that is expressed in skin. The Protein G SepharoseTM 4 Fast Flow beads are pre-incubated with patient sera (or monoclonal against POI). After washing the beads are incubated w/ a substrate containing the POI.

This works fine when we use the MoAb, as it results in clear bands at a proper MW .

When we used patients serum that we know it contains IgG against POI. 60 µl of beads are loaded w/ 10 µl serum. We do get a faint signal. So far the good news. We need a stronger signal though.

We expected adding more serum to the beads would result in a better signal, but to our surprise that did not happen. The signal goes down after adding more patients serum!

We also tried mixing in the good-working MoAb w/ various concentrations of (normal healthy) serum. This resulted in a similar result: the POI signal went down after adding more NHS.

The IgG heavy and light bands do get higher in the IP’s w/ increasing amounts of serum. So it is not that the more serum IgG added is not bound to the beads.

Do you have any idea how to counter this problem?

Figure: POI is indicated w/ arrow. Arrowhead: reduced IgG heavy chain. A fixed volume (5 µl) MoAb is mixed with various volumes normal serum (NS). From a similar experiment the result is shown of IP w 10 µl patient serum (PS).