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Pierce Far-Western Kits FAQ


Answers to frequently asked questions (FAQs) about Pierce Far-Western Kits


 

Q. What types of Pierce Protein:Protein Interaction (PPI) Kits are available?
Q. What methods can be used to detect the prey protein?
Q. Why do the membrane and in-gel far-Western procedures require such different dilutions of the anti-GST antibody?
Q. Can one really look for protein interactions in a denaturing gel or on a membrane?
Q. Are all of the conditions for binding the prey protein to the bait protein described in the kits?
Q. How many far-Western or in-gel detections can be performed using one kit?
Q. How is the tagged protein detected in the gel or on the membrane?
Q. If my protein is endogenously expressed, which kit should I choose?

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. What methods can be used to detect the prey protein?
The prey protein can be detected in-gel (probing targets directly in the elecrophoresis gel) or following transfer on a membrane. Detection on the membrane will be more sensitive, as long as the prey protein transfers easily from the gel to the membrane.

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Q. Why do the membrane and in-gel far-Western procedures such different dilutions of the anti-GST antibody?
The membrane procedures uses 1:100,000 - 1:500,000 dilution of the anti-GST antibody while the in-gel procedures uses 1:2,500 - 1:5,000 dilution of the same antibody. This difference is necessary because protein detection on a membrane is more sensitive than in-gel detection. Since the membrane detection is more sensitive, less HRP-conjugated binding protein (streptavidin or antibody) is required to produce a strong chemiluminescent signal with the Chemiluminescent Substrate on the membrane than is needed for in-gel detection.

Q. Can one really look for protein interactions in a denaturing gel or on a membrane?
Yes, if the SDS is washed out. The alternative is to run a native gel that lacks SDS and reducing agents.

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 with 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. How many far-Western or in-gel detections can be performed using one kit?
The kits have sufficient materials for 10 mini-gels or membrane detections (1,000 cm2 total).

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Q. How is the tagged protein detected in the gel or on the membrane?
The biotin kit utilizes streptavidin-HRP to detect the biotinylated protein bound to the prey protein in the gel or on the membrane. The GST kit utilizes a polyclonal goat anti-GST antibody for detection of the glutathione-S-transferase tagged fusion protein bound to another protein in-gel or on a membrane.

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 detection protein of the binding pair. For help choosing a biotinylation reagent, refer to the Biotinylation Selection Guide available at our website.

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