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Pierce Protein Interaction Pull-Down Kits FAQ


Answers to frequently asked questions (FAQs) about Pierce Protein Interaction Pull-Down Kits


 

Q. What types of Pierce Protein:Protein Interaction (PPI) Kits are available?
Q. Is there a specific lysis buffer and method of protein collection that is recommended with these kits?
Q. After I perform the pull-down of my prey protein, do I recover it free of the bait protein?
Q. Are all of the conditions for binding the prey protein to the bait protein described in the kits?
Q. What are the binding capacities for the affinity supports in these kits and how many pull-downs can be performed per kit?
Q. If my protein is endogenously expressed, which kit should I choose?
Q. How can I determine if my prey protein was successfully pulled down?
 

Q. What types of Pierce Protein:Protein Interaction (PPI) Kits are available?
There are Pull-Down Kits and Far-Western Kits. The Pull-Down Kits are very similar to an immunoprecipitation (IP), but instead of an antibody, a tagged bait protein [biotin (Product No. 21115) , GST (Product No. 21516) or 6xHis (Product No. 21277)] is used to bind the prey or target protein. This protein complex is then purified using an affinity column specific for the tagged bait protein. The Far-Western Kits are similar to Western blots, but again, instead of an antibody, a tagged non-immunoglobulin protein [biotin (Product No. 23500) or GST (Product No. 23505)] is used to bind to the prey or target protein for detection on the membrane.

  Q. Is there a specific lysis buffer and method of protein collection that is recommended with these kits?
Yes. The Pull-Down Kits include a lysis buffer that can be used to obtain both the bait and the prey proteins. The instructions include protocols for lysing bacterial, insect and mammalian cells; working with a previously purified protein; or protein from in vitro transcription/translation reactions.

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Q. After I perform the pull-down of my prey protein, do I recover it free of the bait protein?
This varies with the different kits. The imidazole in the 6xHis Kit and glutathione in the GST elute the tagged bait protein from its support, along with the prey protein. The low pH elution buffer in the biotin kits recovers the prey protein free of bait protein contamination.

Q. Are all of the conditions for binding the prey protein to the bait protein described in the kits?
No. Every protein pair will require unique conditions in order to bind to one another. Factors to consider are pH, ionic strength of the buffer, need for cofactors, whether the interaction is long term or transient, etc. The researcher is recommended to perform a literature search or try several different conditions in order to determine what conditions are best for the binding of their proteins of interest. The kit includes a "universal" wash buffer formed by diluting the lysis buffer 1:1 in TBS.

Q. What are the binding capacities for the affinity supports in these kits and how many pull-downs can be performed per kit?
The kits have sufficient materials for 25 pull-downs and the supports have the following binding capacities:

  1. GST 8 mg GST/ml
  2. 6xHis 12-25 mg 6xHis protein/ml
  3. biotin 9 mg biotinylated BSA/ml

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Q. If my protein is endogenously expressed, which kit should I choose?
We recommend the biotin kit for endogenously expressed proteins. The reason for this is the wide variety of biotinylation reagents that can be used to label the bait protein. For help on choosing a biotinylation reagent, refer to the Biotinylation Selection Guide.

Q. How can I determine if my prey protein was successfully pulled down?
The total protein stains GelCode Blue or Pierce Silver Stain (Product Nos. 24590 and 24612, respectively) can be used to detect proteins in a gel. Alternatively, total protein can be determined on a nitrocellulose membrane using one of the SuperSignal West Chemiluminescent Substrates (Pico-34080, 34079, 34077; Dura-34075; and Femto-34095 and 34094).

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