Did you yourself couple the beads with the antibody? Use a good crosslinker and also give all the recomended washes. Washes are very essential. That might be giving the noise in the negetive controls.  

While Western Blotting, run some amount of your whole cell lysate (for positive protein band) before adding beads. Save all the supernantents to be discarded. Run some amount of the supernatant you get after spinning down the beads (for no/less protein band). These will serve as good controls. Hope this helps. Good luck.

Hello Benan, I don't know the band size of your marker, and I'm not sure to which bands you refer to, but in general, if you use IgGs as negative control, the protein G will pull them down, just like your specific Ab, which is also IgGs... and secondary anti-mouse Abs will recognize them on gel, unless you have a primary Ab for your protein raised in a different species... So, you will always have two very prominent bands at around 20 and 55 kDa, even in an IgG control.

What you have to look for, is the specific band for your IPed protein. If it is the lowest band in the gel you show, it looks like the IP is not working. When IP works properly, the IPed protein should be concentrated (from the signal of the IgG I imagine you are loading probably 10-20% of your IP), so at least equal or stronger signal compared to the Input lane (where you probably load 2-5% of the total protein used for the IP). The fact that the band has same intensity in the IgG control tells you that that is only background signal, unspecific binding to IgGs and not specific IP of your protein by the Ab. So you have to improve the IP conditions.

There are several parameters to look for: salt concentration, detergent type and concentration, pH if the lysis buffer; then protein concentration, temperature, time and incubation strategy in the IP procedure.

While salt concentration might be more important for the co-IP partners, since Ab recognition of the epitope is usually quite independent from salts, detergent type and pH have a strong influence. If the lower band in the WB is your protein of interest, it means your Ab has a high affinity for a denatured protein, which might mean that your epitope might be buried in a native environment. So playing with detergents might help uncover the epitope by partially denaturing the protein. Typically 1% tritonX is used in IPs, but you might want to try to add some stronger detergent, for example 0.2-0.5% Sodium deoxycholate, or 0.1-0.2% SDS (as in RIPA buffers... google it, you'll see there are different formulations), or even a zwitterionic detergent such as 1-2% CHAPS. Moreover typical lysis buffers have pH 7.4 to be close to physiological, but Ab affinity to their epitope is highest above pH 8, which you might want to try. Of course all these manipulations might affect the binding of ligands, so you might find yourself in a dead end situation where either you IP and lose co-IP partners or you cannot IP...

Second part of the problem is how to perform the IP itself. We have observed that IP works most efficiently keeping proteins highly concentrated, so we perform it in minimal volumes of 200-300 ul, containing a total of 0.5-1.5 mg of protein, meaning a protein concentration after lysis of 2.5-5 mg/ml. Secondly, we use 2 ml tubes with a large round bottom for the IP, not the classical 1.5 ml, because they are conical and there no mixing can be obtained using these small volumes. Thirdly tubes are rotated on a wheel at 4 °C, from a minimum of 2 hrs to O/N... of course lysis buffer contains protease inhibitors and often phosphatase inhibitors. Lastly, when adding the proteinA/G beads we dilute the mix to 1 ml with lysis buffer, this decreases sensibly unspecific binding of proteins to the proteinA/G and/or the beads, but does not afect capture of the Ab and IP efficiency. The proteinA/G incubation occurs still at 4 °C for 2-4 hrs on the rotating wheel. 3-4 washes are performed with abundant volume of the buffer (that is normally the same as the lysis buffer but without protease and phosphatase inhibitors), 1-1.5 ml, always at 4 °C and rotating on the wheel for 15-30 min each.

Alternatively to a rotating wheel you can use a gentle shaking on a shaker or orbital shaker/rotator, but you will always need 2 ml tubes to obtain a mixing of the solution which is fundamental.

Good luck.

If I understand correctly, you are suing a Mouse antibody to pull down your protein of interest? Are you using the same antibody for detect the bands on the western blot, or another antibody derived from mouse? If so your secondary antibody may be picking up the heavy and light chain IgG from the antibody used to pull down you protein as well as your control IgG. What is the weight of the protein of interest? Heavy chain IgG is ~50 kDa and light chain IgG is ~ 23 kDa.