Co-IP using magnetic beads, non-specific binding - (Jun/24/2009 )
Hello all, I am using Invitrogen Dynabeads for a coimmunoprecipitation experiment and am getting a high amount of non-specific binding (similar band size on my IgG lane).
The beads are not blocked with BSA due to the hydrophilic nature of the beads surface (as recommended by Invitrogen) but I feel this could be a contributing problem. Does anybody have any suggestions on how to block magnetic beads, or decrease non-specific binding in general with these beads?
fyi - preclearing the lysate with beads seemed to make it worse...
Thanks for your time
You could try higher stringency (i.e. higher salt concentrations, more detergent such as Triton or NP40). It may also alter the binding or your protein to the beads, but you should get cleaner IPs thereafter.
To minimize background binding you can try reducing the incubation time of the IP. 10 min at room temperature is enough for most IPs. You can also try adding detergent to the binding and washing buffer. We recommend Tween-20 to a final concentration of 0.01-0.1%.
A small Protein G may come off the beads during elution, depending on the harshness of the elution: buffer, temperature and time. Eluted Protein G will be visible as two bands on a WB, at approximately 24 and 48 kDa (close to H and L Ig bands). To avoid this, the beads may be washed before use in mild elution buffer (50 mM glycine pH2.7-3), then rinsed in Binding and Washing buffer.
The absolute best beads to use for CoIP are our Dynabeads M-270 Epoxy. The antibody is covalently coupled directly to the beads and the background binding is extremely small. Cristea et.al (2005) describes an excellent CoIP method using Dynabeads M-270 Epoxy: http://bit.ly/1izPn
I hope this is helpful.
I am currently using Dynabeads (magnetic, protein G) from Invitrogen. There was this data here : http://www.invitrogen.com/site/us/en/home/...cipitation.html
and it actually tells us which protein (A or G) suits our work.
I realized there was Total Ig and other Igs (such as IgG2a etc). What is Total Ig? I am sorry if this is silly but the terminology can be a little confusing.
Plus, I am currently using PBST-BSA (0.1%, 0.1%) to wash my beads and 6%BSA-PBS to block the beads. As what the thread started mentioned, the surface is hydrophilic as stated in the product sheet. Is there any other ways of blocking it? I would like to incubate the beads with anti-HA mAb, after which I am performing some washes before I will add in some samples that went through in vitro translation and is supposed to form protein complexes that can be captured by the anti-HA mAb surrounding the protein G beads. Problem is that there is high background noise when I elute the DNA from the beads to carry out real-time PCR.
Would really appreciate some help. THanks in advance!
The 'total Ig' means all the immunoglobulin in each respective species. With three pluses in the table, that bead type will bind a high proportion of the immunoglobulins found in that species. In addition, the table provides information about binding of some of the more common Ig's individually, e.g. IgG.
It sounds like you are isolating a DNA sequence which is bound to a HA-tagged protein? What kind of sample are you isolating this protein-DNA from? What are the lysis conditions and washing conditions? Are you seeing protein background or nucleic acid background? How are you eluting the DNA? Approximately how large is the DNA fragment you want to isolate?
Blocking Dynabeads Protein A or G with BSA in generally not recommended as the beads are hydrophilic, so the BSA will not bind to the bead surface.