I used to have the same problem. I ended up switching from protein G beads to Tosyl-activated magnetic beads (Invitrogen; Dynabeads MyOne Tosylactivated) for covalent conjugation of the antibody to the beads. For this you would also need their magnet which works beautifully. I have had great success!! 

Good Luck!

Using non-reducing conditions (no DTT or BME) works fairly well. There still might be some signal at 50 and 25, but most of it weill be at 80kDa. Of course, some proteins migrate differently under non-reducing conditions, so your proteins might not still be at the same position. Some people have had success with TrueBlot secondaries, which don't recognize denatured antibodies. The best is often to get another primary antibody from a different host.

Although trublot is the best alternative but you may want to adapt to "protein G-beads-linker-antibody " protocols that helps in fixing the antibody in "open orientation" for effective antigen binding. With this system you can use any elute buffer (antibodies will be fixed on beads and thus  they will not be eluted as contaminants). All the best

Following three suggestions:

• low pH glycine elution
• veriblot secondary Ab
• probe with Ab, strip, reprobe with loading control and strip again n reprobe with previously used dilution of you Ab of interest and also reuse 2ndary Ab      Good-luck

Hi Esperanza,

I have had the same problem with my proteins. I could not solve the issue with omiting BME or using native conditions as the protein I was using would get stuck to the beads for some reason. One way was to crosslink the first antibody with the beads. There are many kits for this. The one I used was Pierce Crosslink IP kit. Nevertheless, despite expecting 100%  of Ab retained after elution, I still had minor elution of antibodies when I eluted my protein in some cases (even after washing extensively...).  I also did a control using the same conditions but with a sample lacking the protein of interest (if you can do that, like a KO strain... not sure what type of sample you are using) to make sure that not too much antibody was leaking. Usually this technique really helps you solve the problem. However using antibodies from different sources would be less time consuming as you do not have to prepare the beads, crosslink them and find the best conditions for your IP.

I hope it helps. Good luck with this !

I used to have the same problem. I ended up switching from protein G beads to Tosyl-activated magnetic beads (Invitrogen; Dynabeads MyOne Tosylactivated) for covalent conjugation of the antibody to the beads. For this you would also need their magnet which works beautifully. I have had great success!! 

Good Luck!

I would suggest that you use protein-G-HRP as a detection reagent. This will only react with your primary antibody and not with the denatured heavy and light chains from your IPing antibody. In my hands this is also much more sensitive than the trueblot system, and probably also cheaper (see e.g. http://www.lifetechnologies.com/order/catalog/product/101223). I agree with the above comments about crosslinking - however, even using crosslinking, there can be free heavy and light chain (as well as crosslinked high molecular weight species) that will confound your analysis. A good method would be crosslinking followed by analysis using protein-G-HRP. There is also the option of protein-A-HRP, if this is more suitable for your primary antibody. If you do not like this option, there's also a very useful commercial kit available for linking HRP directly to your primary antibody: Lightning link, by Innova Biosciences. Best of luck!

Hi Gisela. Do you use the protocol which comes with the beads? If not, could you please post your protocol! I would really appreciate it.Thanks Nisha

Hi Nisha,

I do use the protocol that comes with the beads to conjugate my antibody to the beads with just a couple of small changes. 1. Sometimes I'm not able to be at 40 ug antibody/ mg of beads but it seems to be fine at a lower amount of antibody. 2. After incubation with the antibody I block remaining sites on the beads with 0.2 M Tris, pH 8.8 for 4 hrs at 37C instead of using PBS/BSA. This is helpful if you want to sequence the proteins after IP-ing them (no contaminating BSA).

When I do the actual IP I do it in the lysis buffer (20 mM Tris pH7.5, 2 mM EDTA, 137 mM NaCl, 0.1-0.5% Triton X-100, and freshly added, 1 ug/ml each of leupeptin and aprotinin, 0.2 mM PMSF, 0.4 mM sodium-orthovanadate) for about 20-30 minutes then I wash with lysis buffer but without the added protease inhibitors. To elute the proteins I use 0.1 M Sodium-citrate, pH 3.0. I usually elute in very small volumes, like 2 x 10ul or 2 x 20 ul to get concentrated eluates.

NOTE i: Avoid any DTT or mercaptoethanol while doing the IP because those will dissociate the light chains from the heavy chains of your IP-antibody and then you will have the problem of the light chains showing up on your western blot!! So, omit DTT/mercaptoethanol in the lysis buffer when preparing your cell lysates.

NOTE ii: I always do my IP's on freshly lysed cells rather than freezing samples and then using them. 

Hope this helps.

Good Luck!

Gisela