I am troubled by the same issue

Hi,

if I understand correctly, you have microsomes, to which you bind polyclonal antibodies and to those antibodies you bind the Dynabead with Protein G, is that correct?

When you check the purified proteins by SDS-PAGE, do you see the polyclonal antibodies there?

Something prevents interaction between protein G and antibodies or Protein G is being saturated due to the non-specific binding...What are the protocol details (used buffers for equilibration, binding, washing, and elution contents) and is there any composition in your sample prior to immunoprecipitation that may affect the potential binding...

Hi,

sorry to hear about the challenges. below are some general tips to consider

Amount of Ab used

• The binding efficiency dependents on the bead used and the Ab subclass, e.g. IgG1 will bind efficiently to Prot G but to less extent to Prot A beads. When incubating your Dynabeads™ coated with Ab with the lysate containing the target one has to optimize that as this will depend on the primary Abs affinity for the Ag.
• The elution efficiency depends on the volume. Increased volume results in higher elution efficiency. e.g. increasing the elution volume from 20 µL to 100 µL increased elution efficiency by 20 % in in-house experiments. However, this needs to be optimized for each immunoprecipitation.
• Amount of Prim Ab. I would recommend titrating this. In general, between 1-10 µg Ab per 50 µL Dynabeads™ (per IP). The yield of target will depend on how good your prim Ab is.

Low 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. If the target protein is dilute or has a low binding efficiency to the antibody the protein binding time might be extended to 1 hr or longer,
• Try another antibody.

Non-specific 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.
• 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.

kind regards

ketil

Tomáš Hluska

Dear Tomáš Hluska,

Thank you for taking the time to respond! I really appreciate your input. You understood my situation correctly. My aim is to conjugate antibodies with Dynabeads containing protein G and utilize them to capture the target membrane protein antigen present in microsomes.

I apologize for the incorrect information in my previous message. I want to clarify that the design of my antibody involves a short peptide of 14 amino acids in length, derived from the C-terminal sequence of my target protein. Therefore, it should not be considered polyclonal.

Regarding your inquiry about the purified protein analyzed through SDS-PAGE, I have attached the Western blotting results of my microsomes, the No-binding fraction, and the Elute fraction obtained during the immunoprecipitation process. At this stage, I am unsure which band corresponds to my target protein, as its predicted molecular weight is around 40 kDa. However, as shown in the images, the main band appears at a position of less than 10 kDa, which is puzzling to me.

Regardless of the specific band's location, it is evident from the images that the antibody (Ab1) exhibits varying performance in the two experiments: direct Western blotting with microsomes and Western blotting after immunoprecipitation, despite using the same antibody.

Once again, thank you for your assistance and guidance! I am grateful for any further suggestions or insights you may have.