IP elution off of dynabeads - boiling gives a smear (May/20/2008 )
I have been testing a few different elution methods for a proteomics project I am doing using antibodies cross-linked to Protein G dynabeads. To optimize my elution I have been IPing 100ug lysate in 100 uL volume with 25 uL ab-bead slurry (same concentration as they are sold, I think this is only ~1.25 uL of actual beads). Then eluting each IP by a different method (low pH, chaotropic salt, etc).
As a control I was going to simply boil the beads in 2x LSB for 5-10min after washing them, for the elution tests I still boil the beads after elution to see if any protein is left on them. Anyway, whenever I boil the beads, whether right after washing or after an elution, I just get a smear when I run a western on my samples. Sometimes I can still detect my protein in the smear, other times I can't. When I boil sepharose beads under the exact same conditions I get no smear and can see my bands. I suspect there may be some degradation, as I'm using some leftover lysates that were frozen at -20 to optimize. But the result with sepharose suggests the dynabeads have something to do with this.
Has anyone else ever experienced this problem with dynabeads and know what the cause is?
Thanks for any help
I suspect the smear is from protein G, or antibody fragments, coming off the dynabeads. If it is antibody, try boiling or 65C heating in sample buffer minus DTT, remove supernatant, then add the DTT and boil. Of the 4 Ab fragments, only one is probably crosslinked and the other three will be removed with denaturant and a reducing agent.
If the smear is from Protein G, you might need to switch to a different x-linking matrix to get clean westerns.
This may well be due to proteins non-specifically bound to the tube being co-eluted with your target! We have seen on several occasions that when we are eluting (f.ex. by heating/boiling in Sample Loading Buffer) in the same tube as we did the bead IP incubation, a substantial smear on the gel results. However, by transfering the bead suspension to a fresh tube prior to elution, this smear is more or less completely eliminated. Could be worth a try... Apparently this problem can also be avoided by eluting at low pH, f.ex. using a Glycine buffer at pH 2.8.
Additionally, I would recommend to keep the incubation time short to minimize non-specific binding to the beads (<30-60 min is often enough using Dynabeads).
I never trust leftover lysates even if i store at -80. I always prepare new samples.
I have used Dynabeads Protein G for years and am really happy with its performance in IP. Especially compared to sepharose it is so much easier and faster to use. I have not come across any other matrix which can even compare to Dynabeads Protein G for IP. In fact, one of the advantages compared to sepharose is that you can transfer the beads to a clean tube before elution, which greatly reduces the presence of proteins unspecifically bound to the tube. It seems some people don't do the tube transfer before elution though, - I would highly recomend this.
Smitity: Did you try changing tubes? Have you had background/ smear for just one Ab or for several? Of course one should always rule out that it isn't the antibody that is showing unspecific binding. Many of the antibodies we have tried for IP do not work even if they work ok for western blots and vice versa.
Here are some further tips to reduce background binding:
* Use more stringent washing buffer for washing.
* Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations between 0.01-0.1.
* If the beads are blocked before precipitation, add identical blocker to the washing buffer.
* Increase the number of washing steps.
* Prolong the washing steps.
* Decrease incubation time (beads and sample).
* Try the indirect method. (Incubate Ab with the lysate, then add Dynabeads Protein G)
* Decrease the antibody concentration.
* A pre-clearing step may be performed to remove molecules that non-specifically bind to the protein A/protein G or the beads themselves.
Huckle: Elution using low pH is co-called mild or non-denaturing elution. Using mild elution buffers have no direct effect on reducing background although they are of course useful for other reasons :-)
We normally boil (95 degrees) our samples in WB sample buffer with DTT for 5 min and we have not seen any protein G fragments in our eluates. In any case, I very much doubt that these fragment could cause a smear on the gel... Protein G subunits have a Mw 17 kDa and would give a distinct band in that size range on a SDS-PAGE gel.