Hi Robert

Yes this will sort your problem as using rabbit origin antibodies downstream of mouse origin antibodies do give these multiple bands. Room temperature elution will help you in limiting the non specific bands.

Regards

Interesting.

We have had similar problems in the past using Streptavidin-270 dynabeads. We always assumed those bands were IgGs as well. We also found that those beads disappeared when we increased elution stringency during bead preparation.

I have two suggestions for you.

1. Do an experiment where you boil your beads first (like a pre-elution before you incubate with your samples), then do your IP and your real elution (boil again and apply to western blot). See if the bands dissapear then. This worked in our case, but then again we had antibodies attached to the beads and we don't actualy boil the beads.

This brings me to my second suggestion:

2. Do a less stringent elution. We elute normally in Glycin or citric acid (pH

Hello again!

Thanks for confirming our suspicions, Mr Gambhir. Howeve,r we don't see the point why A using rabbit origin antibodies downstream of mouse origin antibodies  gives multiple bands.... We talked with the Commercial Assitance and they told us:

When protein A/G is denatured some binding sites come available that are recognized by the antigen binding site of rabbit antibodies. This is only observed with rabbit and not with other species.

So the solution not to detect denatured protein A/G with rabbit secondary is not to boil.

Strange, anyway. Protein A/G open spaces for...just...rabbit antibodies? Moreover, protein G we think is smaller than 50kb...

We will try to boil the beads, but maybe protein G will separate from the beads and NO antibody will bind them...Thanks for your suggestions, Mr Streng. We'll compare some elutions with Glycin. About crosslinking the antibodies, we also used 270 dynabeads...but we had no better results. Therefore, we changed to an indirect IP, rathen than direct IP preparing crosslinked beads with antibodies.

Thanks for all the answers. I think more people have had this problema but everyone thinks they are the high chain of antibodies, not something about beads...

We also always face the problem of unspecific bands from antibodies during IPs. I am not sure if this would help, but this what I did to eliminate unspecific bands (atleast most of them). Our elution buffer included DTT and I excluded the same, which resulted in a single antibody band (about 98 Kda in my studies). I am also continuously trying to modify my IP in order to get a clearer gel.

Again, I have never tried to elute the beads alone and load it on a gel. The only reason (as mentioned above) could be that Protein G itself is getting eluted.

Hi, have you tested the TrueBlot Ultra Ig HRP secondary? very useful if your target is the same size as heavy or light chain. using this secondary Ab heavy or light chain will not be recognized on the blot.

By the way, of you have any questions regarding Dynabeads feel free to contact me at [email protected] as I am working as a staff scientist at the Dynabeads production facility in Oslo, Norway

Despite the question being quite old, I would like to add to it a few things. I performed an IP with two antibodies (from goat and rabbit) against my protein of interest and included two IgG controls (goat IgG and rabbit IgG). In addition, ath the end of my IP, after my protein had been eluted; I boiled the beads and together with my samples loaded them on a gel. Western Blot was performed using a donkey anti-goat secondary antibody.

My results indicated that a thick band of around 50 kDa is only visible with samples that were incubated with rabbit IgGs. The ones that were incubated with goat IgGs did not have such a band (see file attached), confirming that rabbit IgGs interfere with signal detection.

In case anyone else comes across this question I'd like to add an updated answer:

According to Thermo, recombinant protein A/G can leech off magnetic beads when boiled. Denatured protein A/G can still bind to the Fc region of rabbit IgG and therefore will produce an artifactual band at 40 - 50 kD on SDS-PAGE whether you use a primary OR secondary antibody produced in rabbit. Therefore it is recommend that samples be eluted at room temperature when using protein A/G beads if rabbit IgG will be used for downstream applications (they haven't tested 37C).

I haven't seen data about whether this holds for protein G magnetic beads however the manufacturer's instructions recommend performing the elution at 70C which may suggest that boiling at 95C+ can also cause this artifact with protein G beads.

Hi Christopher,

Have you tried using TrueBlot Secondary antibodies for you IP-western blot? It will help most probably.

Agree with Rajni Kumari however if the issue is that eluted protein G is binding to the Fc portion of your native secondary antibody then this will not fix the problem. I do suspect that boiling the beads is causing elution of protein G which may be able to bind to rabbit IgG. You can wash your beads in PBS then heat them at increasing temperatures to see if protein G is eluting off simply by running a silver stain. Would recommend RT, 37C 50C 75C and 95C for 10 minute in PBS and running the flowthrough on a Bis-Tris gel followed by silver staining with the Termo silver stain kit.

We also recently observed that protein G is detached from the beads (Dynabeads from Thermo) when we perform the elution with 1Xlaemmli (5min at 95°C). It is clear that this is protein G since we get the same band when just boiling the beads without anything else. I called Thermo and they said this shouldn't happen because protein G is covalently linked to the beads... we are trying right now to test different batches and sources of proteinG-coupled to magnetic beads. I hope that this batch-dependent because in our hand, elution with Laemmli is much more efficient than with other methods in particular those using a low pH buffer