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ChromoTek:5 Tips for better immunoprecipitation (IP)!
lebo / 2016-07-21
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Pull-down of proteins can be difficult, particularly when they are expressed at low levels. Here we present 5 tips, which will considerably improve your IP results.

1.    Protein concentration matters
The higher the protein concentration, the higher the IP yield. It makes a significant difference if the concentration of your protein during IP is 1 nM (low protein concentration) or 1000 nM (high protein concentration). Try to use a concentration as high as possible.

2.    Binding affinity matters
The higher the binding affinity, the higher the protein yield. A specific antibody, which works well using high abundant proteins, may perform poor when low abundant proteins shall be precipitated. Make sure that your antibody has a low dissociation constant KD respectively high affinity. Otherwise your antibody binds just a very small fraction of your protein. For instance, the ChromoTek GFP-Trap has a very low KD of 1 pM (high affinity) and enables very efficient pull-downs of very low protein concentrations.
 
Same protein input, different KD affinity matrices resulting in different yields:

3.    Time matters
The shorter the wash time, the better the pull-down yield. The optimal wash time has to be determined experimentally (also consider background). However, the use of affinity media with low off rates helps to avoid leaching of your bound protein.

4.    Specificity matters
The better the specificity of the affinity matrix, the lower the background. Use only specific, well characterized, and validated affinity matrices to obtain reliable, reproducible results.

5.    Volume matters
The smaller the volume, the more effective your IP works. Using a small volume keeps your protein concentration high and therefore increases the binding affinity.

For a detailed explanation and description, download our white paper here:

 

Here we demonstrate the superior performance of the ChromoTek Nano-Traps affinity media using GST-Trap and MBP-Trap and present a theoretical framework how their low dissociation constants translate into effective immunoprecipitation of proteins expressed at low levels or from dilute solutions.

 

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