Immunoprecipitation (IP) is a valuable tool for studying protein-protein interactions across various fields. And though the procedure involves a simple, well-established protocol, generating accurate, reliable, and repeatable results requires attention to key details. Reagents with rapid binding kinetics and low nonspecific binding are critical for high yields and precise data.
InvitrogenTM DynabeadsTM magnetic beads are nonporous, uniform, superparamagnetic, monodispersed polystyrene beads widely used for immunoprecipitation and regarded as a “gold standard” reagent. Their uniform structure creates consistent physical and chemical properties, which have many benefits:
- low non-specific binding
- high reproducibility
- fast binding kinetics
- low antibody consumption
Dynabeads are also automation-ready, reducing hands-on time and increasing throughput. To help get the most out of automation and immunoprecipitation and to answer some of the technical questions we get commonly, our technical experts conducted several experiments regarding various parameters of the automated Dynabead protocol.1
Does Antibody Incubation Time Affect Binding Efficiency?
Not beyond about 10 minutes. Due to Dynabeads’ rapid binding kinetics, near-complete binding is achieved rapidly, and extending incubation beyond that period does not increase yield. We tested this by using a chemiluminescence assay to monitor IgG1 binding to Dynabeads Protein G on a KingFisherTM Flex instrument, but the same result held up using a manual protocol.
Does Mixing Speed During Wash Steps Affect Binding Efficiency?
Yes. Mixing too vigorously with a fast mixing speed causes loss of antibodies and reduced target yield. We tested slow, medium, and fast mixing conditions. Additionally, chemiluminescence was measured to determine binding efficiency. Compared to slow washing, medium-speed washing led to a 4% loss in conjugated antibodies, and fast-speed washing caused a 32% loss. Slow and medium washing was similar to results using a manual protocol.
Does the Number of Wash Steps Affect Binding Efficiency?
Excessive washing after antibody binding can cause partial loss of bound antibody from the bead’s surface. We used chemiluminescence to measure the loss of mouse IgG1 after 1, 2, or 3 wash steps. Increasing 1 to 2 washes caused an 8% loss of antibody, and adding a third step increased the loss to 12%. A single wash step was sufficient for optimal IP results, and a single manual washing step was comparable to a single automated wash step.
Does the Wash Volume Affect Binding Efficiency?
Not to a significant degree. We performed buffer washes with 100, 200, and 500 μL of buffer with Dynabeads Protein G and measured binding via chemiluminescence. Relative to the 200μL washes, 100 μL volume resulted in just 3% loss of antibody, and 500 μL volume resulted in just 5% loss. Similar results were collected for manual immunoprecipitation protocols.
Is background noise experienced during immunoprecipitation preventable?
Background during immunoprecipitation is caused primarily by two sources. When using a separation method based on slurry solid phase, the lab technician must carefully wash and pipette buffer after each step. When using a slurry, solid-phase method and pipetting, you can accidently removing some of the slurry, and therefore some of your sample, or end up leaving some remaining buffer behind that ultimately contain unwanted protein that gives rise to unwanted background. Porous sepharose and agarose, means a lot of unwanted liquid gets trapped.
Unwanted proteins are therefore trapped within these pores. Even with washing steps, you can end up with these left over proteins and therefore background. Dynabeads use magnetic separation to pull the bound bead pellets to the side of the tube. The surface of the Dynabeads, is also non-porous. This means all binding happens on the outer surface, with no inner surface or cavity to trap any unwanted proteins. Learn more myths about immunoprecipitation that have been debunked.
Does the Binding Temperature Affect Binding Efficiency?
Not really. For most applications, room temperature incubation is likely adequate. After incubation at room temperature or 4°C for 10 minutes, we measured chemiluminescence and found that room temperature incubation resulted in only a 9% loss of antibody compared to 4°C.
Does the Capture Volume Affect Binding Efficiency?
Yes. Researchers should optimize concentration and capture volume for their antibodies. While holding the amount of Dynabeads Protein G magnetic beads constant, we varied the isolation volume from 100 μL to 1 mL. As the volume increased and the concentration reduced, binding efficiency dropped. Using 1 mL capture volume caused a 19% loss in binding compared to 100 μL. Manual protocols using 200 μL capture volumes generated results similar to 100-200 μL capture volumes using automated instrumentation.
Does the Antibody Subclass Affect Binding Efficiency?
Yes. Antibody subclasses vary in their ability to bind their targets. We tested several antibody subclasses with Dynabeads Protein G. The amount of antibody was held constant at 5 μg. Compared to IgG1, all subclasses tested – IgG2a, IgG2b, and polyclonal rabbit IgG – showed reduced binding efficiency, 55%, 79%, and 62%, respectively. These differences would significantly impact performance.
Does the Elution Volume Affect Release Efficiency?
Yes. Dynabeads Protein G was coated with primary antibody against CD9 and incubated with HeLa cell lysate to test this. Elution volumes of either 20 or 100 μL were tested, and we found that increasing from 20 to 100 μL elution volume increased percent eluted by approximately 20%. When well size allows, increasing elution volume increases release efficiency.
Conclusion
Combining Dynabeads’ exceptional uniformity with the precision and speed of automation can help you get better results faster in your IP. Careful optimization of any procedure is crucial, and the data presented here should help you understand the flexibility of various parameters in automated and manual Dynabeads protocols.
To get more answers to your questions about Dynabeads and Thermo Fisher Scientific’s automation solutions, visit our magnetic bead-based immunoprecipitation page.
This article is for Research Use Only. Not for use in diagnostic procedures.
References
- Dynabeads magnetic beads—the key to successful immunoprecipitation. Thermo Fisher Scientific website. Accessed July 1, 2022. Published March 9, 2021.
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