Confocal-based bead-binding

In order to prepare proteins for a fluorescent bead-binding experiment, you need one protein that can bind a resin (in my case MBP-Rubicon RH domain, which can bind amylose resin) and a fluorescently labelled bait protein (in my case, Rab7-AlexaFluor 647).

To prepare my AlexaFluor 647, used an NHS ester salt, and followed standard manufacturer procedures for labelling, quenching, and cleaning the protein (https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/nhs-ester-oligonucleotide-conjugation)

Measure the degree of labelling of your sample. My degree of labelling was 0.9 fluorophores per Rab7 copy

Here, I wanted to assess the impact of phosphorylation on binding. In order to keep the samples as comparable as possible, I fluorescently labelled my Rab7 with 647, then separated the Rab7 into two pools. One pool I phosphorylated using TBK1, the other pool I treated with the same dilution and recovery steps, but omitted the TBK1. See the TBK1 phosphorylation protocol for details.

Washed and prepared a 40 uL suspension of 25% amylose resin in a 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, and 10 mM TCEP buffer. Incubated this resin with 500 nM MBP-RH domain and ~ 4 uM fluorescently-labelled Rab7.

Prepare a negative control consisting of the experimental sample absent the resin-binding partner, in this case MBP-Rubicon RH

Incubate samples on rocker at room temperature for 1 H.

Collect resin at bottom of tube using a tabletop centrifuge, and aspirate and discard supernatant.

Wash resin 3 times with 1 mL of room temperature buffer

Resuspend resin in 200 uL of buffer, then transfer to an 8-well confocal imaging chamber.

Focus sample and image resin. Adjust imaging parameters if necessary.

In order to measure the brightness of an individual bead, export the images to FIJI, and define an ROI that contains a bead edge. Measure this ROI, and record the maximum brightness. This brightness should correspond to the edge of the bead. Repeat this for ~20 cells, and average to acquire a single biological replicate.

Repeat experiment at least twice more to acquire two more biological replicates. Perform a T-test between the comparison groups in the experiment