Troubleshooting and FAQ's for immunoprecipitation with Dynabeads®
Products for Immunoprecipitation:
- I seem to get low binding of my selecting antibody with Dynabeads® Protein A or Protein G. What can I do to improve binding?
Cross-linking / Covalent coupling FAQs:
- I was able to immunoprecipitate my protein using Dynabeads® before, but after cross-linking the antibody to the beads, I get no protein bands on my gel.
- I have cross-linked antibody to the Dynabeads® Protein A or Protein G, but antibodies are still coming off the beads during elution.
- I removed the non-cross-linked antibodies before performing the immunoprecipitation step, but still get bands on gel indicating that the antibody is coming off the Dynabeads®.
Non-Specific Binding & Blocking FAQs:
- As negative control, I have incubated my sample with Dynabeads® Protein G that are not coated with antibody, but I get non-specific binding to the beads.
Other Options for Immunoprecipitation:
- I have used biotinylated antibodies coupled to Dynabeads® Streptavidin for IP. How do I elute my target protein from the antibody without eluting the antibody off the beads?
What is the difference between Dynabeads® Protein G and Dynabeads® Protein A?
- Dynabeads® Protein G and Dynabeads® Protein A are 2.8 micron beads covalently coupled with Protein G and recombinant Protein A, respectively.
- Protein G and Protein A differ in their binding strength to immunoglobulins from different species and subclasses.
- See the Selection Guide for detailed information on Ig species and subclass specificity to Dynabeads:
I seem to get low binding of my selecting antibody. What can I do to improve binding?
- Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.
- Check the binding of your antibodies to the beads. If the antibodies are not captured and bound to the beads, the immunoprecipitation experiment will not work.
- If you have used the indirect method, try the direct method. Conversely, if you have used the direct method, try the indirect method.
- Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.
- Increase the incubation time.
- Try another antibody.
I was able to immunoprecipitate my protein using Dynabeads® before, but after cross-linking the antibody to the beads I get no protein bands on my gel.
- The binding sites of your antibody has likely been altered by the crosslinking.
- When this occurs your antibody will show reduced affinity or no affinity to its target antigen
- Another consequence of crosslinking can also be increased affinity for unintended (non-specific) targets
- This is always a high risk with crosslinking, and it is a problem easily avoided by choosing another path to covalent antibody coupling.
- Try the Dynabeads® Antibody Coupling Kit
- This kit is a far superior solution for covalent antibody coupling to Dynabeads (compared to crosslinking with Dynabeads Protein A or G).
- The antibody coupling kit is compatible with almost any antibody.
- The coupling kit is designed specifically for covalent antibody coupling to Dynabeads.
- Unlike crosslinking, the Dynabeads® Antibody Coupling Kit will not alter antibody specificity or affinity.
I have cross-linked my antibody to the Dynabeads®, but there are still antibodies coming off the beads during elution.
- Cross-linking will never be 100%. Some antibodies are not cross-linked and may come off under elution.
- Perform a washing step with low pH directly after cross-linking to remove non-cross-linked antibodies.
- Remember to bring the pH back to normal before IP.
I removed the non-cross-linked antibodies before performing the immunoprecipitation step, but still get bands on gel indicating that the antibody is coming off the Dynabeads®.
- If you are using reducing agents in the sample buffer before gel loading, try a sample buffer without reducing agents.
- Reducing agents such as DTT or ß-mercaptoethanol will break disulfide bridges within antibodies resulting in release of the antibody light and heavy chains.
- Another option is to elute the protein by lowering the pH, this should leave the antibody bound to the beads.
I experience non-specific binding in my immunoprecipitation experiment.
- 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.
- 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.
Are Dynabeads® Protein A and Dynabeads® Protein G pre-blocked with BSA?
- No, Dynabeads Dynabeads® Protein A and Dynabeads® Protein G are not pre-blocked with BSA
How can I block Dynabeads® Protein A and Protein G with BSA?
- Blocking with BSA works best on a hydrophobic surface, where surface adsorption keeps the blocker in position.
- Dynabeads® Protein A and Protein G have a hydrophilic surface.
- Hence, BSA blocking may not be very successful, as surface adsorption is not promoted between the hydrophilic bead surface and the hydrophobic BSA.
- Instead, reduce non-specific binding by performing more stringent washing and by adding Tween-20 detergent (concentrations of 0.01-0.1% ) to the washing buffer.
As negative control, I have incubated my sample with Dynabeads® Protein G that are not coated with antibody. When I do this I get non-specific binding to the beads themselves.
- Using non-antibody-coated Dynabeads® Protein G (or Dynabeads® Protein A) with your IP sample is not a good control.
- Different molecules in your sample will bind either to protein G or to the beads themselves through a variety of interactions
- Hydrophobic interactions, charge interactions etc).
- A better negative control would be to use Dynabeads® Protein G bound to an irrelevant IgG.
- If you truly want the lowest possible levels of background binding, use the Dynabeads® Antibody Coupling Kit.
- The beads provided in this kit have ultra ultra low levels of background binding.
- And since your antibodies will be covalently coupled to the bead surface, the antibodies will not come off the bead surface when you elute your pure target protein, giving an elutate that is extremely pure for your target protein.
I have used biotinylated antibodies coupled to Dynabeads® Streptavidin for immunoprecipitation. How do I elute my target protein from the antibody without eluting the antibody off the beads?
- Use mild elution conditions, e.g., a buffer with high salt or low pH.
- Do not heat the beads at 95°C for 5 minutes in SDS buffer as this will elute the antibody as well as your target protein.
Can I use other Dynabeads® for immunoprecipitation?
- Yes, see selection guide
- Dynabeads that may be used for immunoprecipitation:
- Dynabeads Antibody Coupling Kit
- Dynabeads Co-Immunoprecipitation Kit
- Dynabeads pre-coated in Sheep α-mouse IgG (use with mouse 1° antibodies)
- Dynabeads pre-coated in Sheep α-rabbit IgG (use with rabbit 1° antibodies)
Can I use larger (4.5 micron) Dynabeads® for immunoprecipitation?
- Yes, you can use the larger beads.
- You can use Dynabeads® Sheep anti-Rat (Cat. #: 11035) when your primary antibodies are of rat origin.
- Note: The smaller beads provide larger surface area and therefore give higher yields of protein than the larger 4.5 micron beads.