The pull-down of proteins is an established technology. It can be difficult, particularly when the protein of interest is expressed at low levels. Here we discuss 5 tips that help you to improve your IP results.
Protein concentration matters
Generally, the concentration of the protein of interest is unknown when conducting an immunoprecipitation; however, the yield of an IP does depend on that protein’s concentration: the higher the protein concentration the higher the IP yield. We estimate the intracellular protein concentration equals about 1 nM for low expression level, about 50 nM for endogenous protein expression level, and about 1,000 nM for high expressed respectively overexpressed proteins; dependent on cell type.
Binding affinity matters
A specific antibody, which effectively pulls down a high abundant protein, may not work well when immunoprecipitating that protein at low concentration. Make sure that your antibody has a low dissociation constant KD respectively high affinity because low affinity antibodies bind to only a very small protein fraction. The higher the binding affinity of the IP resin the higher the protein yield. Example: At a protein concentration of 50 nM, a low affinity media IP resin with a KD of 150 nM binds only 25% of the protein, whereas a high affinity resin with a KD of 1nM binds almost all, i.e. 98% of the protein; based on calculations. However, the ChromoTek GFP-Trap has a remarkable low KD of just 1 pM (ultra-high affinity) and therefore enables efficient pull-downs of even low protein concentrations.
The optimal wash time must be determined experimentally. Effective binding of the protein of interest (for Co-IPs the formation of the protein complex), protein degradation over time, and removal of background by multiple washing steps need to be considered. Use high affinity IP resins when the protocol requires harsh and prolonged washing steps. However, the use of IP resin with low dissociation- or off-rates helps to avoid leaching of your bound protein. Using an IP resin with high binding- or on-rates shortens incubation times and is in general gentler to your protein of interest. The shorter the wash time the better the pull-down yield.
The better the specificity of the affinity matrix the lower the background. Use only specific, well characterized, and validated IP resins to obtain reliable, reproducible results.
The smaller the volume, the more effective your IP works. Using a small volume keeps your protein concentration high and therefore increases the binding yield. Concentration is a function of volume. Try to use a volume as small as possible. That keeps your protein concentration high and therefore increases the binding efficiency.
In summary, we recommend keeping protein concentration, binding affinity, processing time, IP resin’s specificity, and buffer volume in mind when planning your next immunoprecipitation to obtain the best immunoprecipitation results.
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Why is nanobody immunoprecipitation different to antibody immunoprecipitation? How does it work?
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