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Pierce™ His Protein Interaction Pull-Down Kit - FAQs

Pierce™ His Protein Interaction Pull-Down Kit - FAQs

查看更多产品信息 Pierce™ His Protein Interaction Pull-Down Kit - FAQs (21277)

6 个常见问题解答

If my protein is endogenously expressed, which Thermo Scientific Protein:Protein Interaction (PPI) 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How is the tagged protein detected on the membrane using the Thermo Scientific Protein:Protein Interaction (PPI) kits?

The biotin kit utilizes streptavidin-HRP to detect the biotinylated protein bound to the prey protein 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 on a membrane.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How many reactions can be performed using one Thermo Scientific Protein:Protein Interaction (PPI) kit?

The Thermo Scientific Pull-Down Protein:Protein Interaction Kits contain a complete, validated set of reagents specifically developed for performing pull-down assays.The kits contain sufficient materials for conducting 25 pull-down assays.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Are all of the conditions for binding the prey protein to the bait protein described in the Thermo Scientific Protein:Protein Interaction (PPI) 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

With the Thermo Scientific Protein:Protein Interaction (PPI) kits, what methods can be used to detect the prey protein?

The prey protein can be detected following transfer on a membrane. The membrane procedures uses 1:100,000 - 1:500,000 dilution of the anti-GST antibody.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What types of Thermo Scientific 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 (Cat. No. 21115) , GST (Cat. No. 21516) or 6xHis (Cat. 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 (Cat. No. 23500) or GST (Cat. No. 23505)] is used to bind to the prey or target protein for detection on the membrane.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.