Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

This method can help us answer key questions in cell biology and biochemistry, such as how the dynamic assembly of protein and protein complexes control signaling pathways and cell behavior. The main advantage of this technique is that we can specifically isolate two interacting proteins, while excluding uncomplexed individual proteins, and competing binding partners. Though we use this method to provide insight into receptive tyrosine kinase dimerization in breast cancer, it can also be applied to any other physiological setting or disease.

We first had an idea for this method when we realized the GFP nanobody recognized the three-dimensional epitope of GFP and other fluorescent proteins. To begin, add 150 nanograms of entry vector, 150 nanograms of destination vector, and eight microliters of TE buffer, to a 0.2 milliliter tube. Next add two microliters of recombinase and ZiMex, and centrifuge the two briefly.

Incubate the reaction for one hour at room temperature, then add one microliter of Proteinase K solution, and incubate at 37 degrees celsius for 10 minutes to stop the reaction. Next, thaw heat shock-competent cells on ice. And transfer 50 microliters of the cells to a 14 milliliter round-bottomed polypropylene tube.

Add one microliter of reaction product to the cells, and mix gently. Incubate the cells for 20 minutes on ice. Then heat shock the cells in a 42 degree celsius water bath for 45 seconds before returning the cells to the ice.

Add one milliliter of LB media to the cells. And incubate the sample with shaking for one hour at 37 degrees celsius. Then, plate the transformation onto a 10 centimeter agar plate with ampicillin, and incubate at 37 degrees celsius overnight.

To begin plasmid purification, add ampicillin and 100 milliliters of LB media to a large conical flask. Then use an inoculation loop to remove a single colony from the agar plate. Submerge the inoculation loop in LB media and briefly mix, then cover the top of the flask with aluminum foil, and incubate it overnight, at 37 degrees celsius with shaking.

From here purify the plasmid DNA using a standard Maxiprep plasmid DNA purification kit, before proceeding with transfection. First, seed HEK 293T cells in 10 centimeter dishes, containing 10 milliliters of DMEM. Then dilute 2.5 micrograms of each bimolecular fluorescence complementation vector in 500 microliters of transfection buffer.

Add 10 microliters of the transfection reagent, and vortex the mixture for 10 seconds. Then briefly centrifuge the samples, and incubate them at room temperature for 10 minutes. Next add the DNA transfection mixture dropwise to the dish.

Then incubate the samples for eight to 24 hours. Prepare a cell lysis buffer and supplemented cell lysis buffer as outlined in the text protocol. Then wash the cells with ice cold PBS twice.

Aspirate the PBS and add one millimeter of ice-cold supplemented cell lysis buffer. Next, place the dish on ice and incubate for five minutes. Then use a cell scraper to remove the cells, and transfer them to a chilled micro-centrifuge tube.

Centrifuge the tube at 18, 000 times gravity for five minutes at four degrees celsius, to remove the cellular debris, then transfer the clear supernatant to a fresh micro-centrifuge tube. Wash an appropriate volume of agarose beads in one milliliter of PBS, then centrifuge the beads at 300 times gravity, and carefully remove the supernatant. Next add 20 microliters of agarose beads to each sample.

And incubate at four degrees celsius for two hours, with end-to-end rotation. Centrifuge the beads at 300 times gravity and wash them in cell lysis buffer three times. Then re-suspend the washed beads in 50 microliters of diluted sample buffer.

Finally, heat the samples at 95 degrees celsius for two to three minutes. In this study, the BiCAP method is used to observe a positive interaction for several protein types approximately 16 hours after co-transfection of HEK293T cells. Con-focal microscopy is used to observe the dimerization of the RTKERBB2 at the plasma membrane, and the binding of Ubiquitin to the E3 ligase UBR5.

After affinity purification with the GFB nanobody, only the full length Venus control protein, and interacting V1 and V2 tagged proteins can be detected in the co-transfection sample. While attempting this procedure, remember to optimize the placement of the bi-molecular fluorescence complementation tag, and either the C-terminal or N-terminal end of your protein of interest. This is necessary to observe a positive protein/protein interaction.

After watching this video you should have a good understanding of how to perform the BiCAP technique, and how to apply it to your protein interaction of interest. Following this procedure, many of the downstream characterization techniques could be employed, like ChIPseek to answer additional questions, such as how dimerization of the transcription factors controls DNA binding.