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Using Purekine "fishing gear" to fish, the protein club "will be on the bait"

Date:2020-10-23 Views:460

In the last issue, the editor brought you "Dry Goods|Fishing with IP "Hooks", Protein Club "Wishers Take the Bite", and the response was very enthusiastic. In the voice of everyone, the editor is rushing through the previous experimental notes, bringing you the second bullet of protein interaction knowledge-Pull-Down. If the protein interaction is compared to an interesting "fishing" activity, then the Purekine filler we use is a tough and slender "fishing rod" and "fishing line". The known GST-labeled recombinant protein is sharp And the perfect combination of strong "fish hook" and "fish bait", and our target protein is the "fish" that moves freely in the water.

The concept of Pull-Down is similar to Co-IP, and its purpose is to study proteins or ligands that bind to known bait proteins. Pull-Down aims to prove the interaction between two proteins or explore unknown proteins or molecules that can bind to the target protein. Pull-Down is different from IP or Co-IP in that it is not based on antibody-antigen interaction, not an immune response. The bait protein (or ligand) is immobilized on a solid support through a non-antibody affinity system. This immobilization can be covalently coupled to the activated microbeads, or through the receptor molecule on the support The bound affinity tag binds and thus immobilizes. For example, immobilized metal chelating affinity chromatography (IMAC) resin can be used to pull-down histidine tag bait protein. The optimization of Pull-Down needs to consider the special nature of the affinity system used. The principle of Pull-Down can be understood at a glance according to the following diagram:

 

The analysis method of the captured protein obtained by Pull-Down is shown in the figure below:

Comparison of Pull-Down and protein purification experiments:

  1. Use recombinant gene expression protein, and then perform downstream protein extraction and purification experiments:

  1. Analysis of the similarities and differences between the two experimental steps:
Experiment type Pull-Down protein purification
Solid carrier Affinity chromatography resin Affinity chromatography resin filler
Carrier form In bulk Column
Incubation method Shaker incubation Incubate in the purification column
buffer Binding Buffer

Wash Buffer

Elution Buffer

Binding Buffer

Wash Buffer

Elution Buffer

Target protein form Prey protein Recombinant protein
  1. Types of products to be tested:
Product Category Cat.
His Tag protein purification tool BMR20000
GST Tag protein purification tool BMR20100
MBP Tag protein purification tool BMR20206
Flag Tag protein purification tool BMR21504
Biotin Tag protein purification tool BMR20306
Protein A Antibody purification tool BMR21604
Protein G Antibody purification tool BMR20600
Protein A/G Antibody purification tool BMR20704
Protein L Antibody purification tool BMR20800