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Increased signal in IP after peptide addition? - Immunoprecipitation signal amplification (Sep/04/2006 )

Hi there,

we are immunoprecipitating an HA-tagged protein, and found a grealty increased IP signal after addition of peptide to the IP - which I cannot explain, and would love to know the reason of.

The IP-protocol was as follows:

- IP on membrane fraction of either WT-HEK cells, or HEK-cells overexpressing an HA-tagged protein.
- Additon of the HA-antibody overnight
- Addition of beads the following morning for 2 hours to IP's in WT and HA-expressing cells. Addition of beads and HA-peptide to HA-expressing cells only for same duration. (I do know that as a control for unspecific binding the antibody should have been pre-incubated with the beads, but it was done in the wrong order. Anyways, another person in the lab found the same effect of an increased signal in the presence of a peptide after IPing with an antibody pre-incubated in the blocking peptide.)
- 3 Washes of the beads
- Elution using Laemmli Buffer (incl. DTT & SDS)
- 8% Tris-Glycine gel and blotting onto PVDF (Hybond / Amersham)
- probing with an biotinylated HA-antibody.

There is no IP in the WT-cells (as expected), and a little IP-signal in the HA-expressing cells. But the IP in presence of the antibody just gives the hugest IP-signal I have ever seen, with the specific bands being stronger than in the lysate control (lane with just the lysate used for the IP). As a shame we haven't got a band for the IP in the presence of the peptide in WT cells.

Has anybody ever experienced such an enhancement of an IP-signal after peptide addition? Does anybody have an explanation for this phenomenon?

Thanks,
Katja

-tardigrada-

QUOTE (tardigrada @ Sep 4 2006, 01:26 PM)
Hi there,

we are immunoprecipitating an HA-tagged protein, and found a grealty increased IP signal after addition of peptide to the IP - which I cannot explain, and would love to know the reason of.

The IP-protocol was as follows:

- IP on membrane fraction of either WT-HEK cells, or HEK-cells overexpressing an HA-tagged protein.
- Additon of the HA-antibody overnight
- Addition of beads the following morning for 2 hours to IP's in WT and HA-expressing cells. Addition of beads and HA-peptide to HA-expressing cells only for same duration. (I do know that as a control for unspecific binding the antibody should have been pre-incubated with the beads, but it was done in the wrong order. Anyways, another person in the lab found the same effect of an increased signal in the presence of a peptide after IPing with an antibody pre-incubated in the blocking peptide.)
- 3 Washes of the beads
- Elution using Laemmli Buffer (incl. DTT & SDS)
- 8% Tris-Glycine gel and blotting onto PVDF (Hybond / Amersham)
- probing with an biotinylated HA-antibody.

There is no IP in the WT-cells (as expected), and a little IP-signal in the HA-expressing cells. But the IP in presence of the antibody just gives the hugest IP-signal I have ever seen, with the specific bands being stronger than in the lysate control (lane with just the lysate used for the IP). As a shame we haven't got a band for the IP in the presence of the peptide in WT cells.

Has anybody ever experienced such an enhancement of an IP-signal after peptide addition? Does anybody have an explanation for this phenomenon?

Thanks,
Katja



First one thing: it is better to incubate your lysate with the Ab and then with the beads, because if you incubate Ab and beads and you try to isolate large protein complexes, you could have problems, because the Ab-beads could act as a kind of resin and your complexes would be too big to access the Ab "packed" in this kind of resin...

Let me understand: you did your IP and detected a strong signal where you used an anti-HA Ab in a lysate containing an HA-tagged protein; you did the same adding a competing peptide (HA peptide) and you saw a lower signal... Did I understand correctly? If so, where is the problem? This peptide competes with your protein for the binding to the Ab, therefore it is logical that you IP less protein. This is the technique people use to elute ptoteins without Ab carryover...

-dnafactory-

your HA-peptide appears to bind some proteins of lysate. To check the binding potency of HA-peptide to your lysate or membranes, simply try a far-like western with your HA-peptide:
-Separate your total lysate in SDS-PAGE in at least 2 slots and blot
-"overlay" one blot with HA-Ab, the other with HA-peptide preincubated HA-Ab (direct method)
- check signal;
if your peptide has binding potency to some proteins of your lysate, you will find additional bands on the blot with the peptide preincubated antibody

immobilize your peptide and clear lysate;

-The Bearer-