Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

The overall goal of this tandem affinity purification method is to isolate protein complexes expressed from eukaryotic cells for biochemical characterization and molecular assemblies and identification of novel interactors and post-translational modifications that regulate their function. This method makes possible the purification of intact protein complexes from eukaryotic cells, to identify novel interactors and regulatory post translational modifications that fine-tune their function. The main advantage of this technique is that it allows for high purity and yield of protein complexes for downstream in vitro applications to study their function and activity.

Cells are collected from STREP affinity purification 48 hours post transfection. Remove the media and add eight milliliters of cold PBS. Pipette up and down to detach the cells from the plate.

Collect and transfer the cell suspension into a 15 milliliter tube and spin the cells down at 800 times g for four minutes at four degrees Celsius. Remove the PBS supernatant. Spin down at 800 times g for one minute at four degrees Celsius to eliminate any residual PBS.

To begin this procedure, resuspend each cell pellet in five times the cell pellet volume of passive lysis buffer, or PLB. Transfer the cell suspension to a 1.5 milliliter microcentrifuge tube. Briefly vortex the suspension, and nutate for 30 minutes at four degrees Celsius.

Spin down the cell suspension at 10, 000 times g for 10 minutes at four degrees Celsius. Transfer the soluble lysate into a new 1.5 milliliter microcentrifuge tube and discard the cell pellet. Save 10%of the final volume of the soluble lysate as STREP-AP input and store it at four degrees Celsius.

It is important to use STREP beads for the first affinity purification step because complexes pulled down with STREP beads recover significantly more protein than those pulled down with the FLAG beads. Using a wide mouthed pipette tip remove the appropriate amount of 50%STREP bead slurry for the affinity purification. Spin down at 1, 500 times g for two minutes at four degrees Celsius.

Remove the supernatant and add 500 microliters of PLB to the beads. Nutate the bead mixture for five minutes at four degrees Celsius. Spin down again.

After the third wash, remove the supernatant and resuspend the beads in PLB to a final volume of n times 40 microliters, where n is the number of samples. Add 40 microliters of 50%STREP bead slurry to each cell lysate sample and nutate for two hours at four degrees Celsius. During this protein production and recovery multiple plates of cells can be used, and the same protein samples lysed from different plates can be combined with one portion of beads during the STREP affinity purification.

After the cell lysate and STREP bead slurry mixtures have incubated for two hours, spin them down at 1, 500 times g for two minutes at four degrees Celsius. Save the supernatant as STREP AP flow through and store at four degrees Celsius. Add 500 microliters of STREP AP wash buffer one to the beads.

Nutate the beads in buffer mixture for five minutes at four degrees Celsius and centrifuge at 1, 500 times g for two minutes at four degrees Celsius. Discard the supernatant and wash again with 500 microliters of STREP AP wash buffer one. After the third wash, transfer the bead solution to a new 1.5 milliliter microcentrifuge tube to reduce the protein background.

Wash a fourth time with STREP AP wash buffer one. After the spin, remove the supernatant and add 500 microliters of wash butter two. Equilibrate the beads by inverting the tubes and then spin down at 1, 500 times g for two minutes at four degrees Celsius.

Discard the supernatant. Elute the protein of interest with 50 microliters of STREP elution buffer. Vortex at 800 rpm for 15 minutes at four degrees Celsius.

Spin down the beads at 1, 500 times g for one minute at four degrees Celsius. Collect the supernatant. This fraction corresponds to the STREP AP elution sample.

Save the beads fraction at four degrees Celsius for a second elution. Aliquot 10%of the STREP AP elution for silver staining and/or Western blotting. To increase the amount of starting material for the subsequent FLAG immunoprecipitation the first and second STREP elutions can be pooled together for more efficient recovery of the FLAG elution.

Begin this procedure by using a wide mouthed pipette tip to transfer the appropriate amount of FLAG bead solution to a microcentrifuge tube. Spin down at 1, 500 times g for two minutes at four degrees Celsius. Remove the supernatant and add 500 microliters of FLAG wash buffer to the beads.

Spin down at 1, 500 times g for two minutes at four degrees Celsius. Discard the supernatant and wash again with FLAG wash buffer. After the third wash, remove the supernatant and resuspend the beads in FLAG wash buffer to a final volume of n times 16 microliters, where n is the number of samples.

Add 16 microliters of the FLAG bead slurry to the remaining STREP AP elution and nutate overnight at four degrees Celsius. Following an overnight incubation of the FLAG bead slurry and STREP AP elution, proceed with FLAG immunoprecipitation. Spin down the FLAG bead suspension at 1, 500 times g for two minutes at four degrees Celsius.

Save the supernatant as the FLAG IP flow through and store at four degrees Celsius. Add 500 microliters of FLAG wash buffer to the beads. Nutate for five minutes at four degrees Celsius and centrifuge at 1, 500 times g for two minutes at four degrees Celsius.

Discard the supernatant and wash again with 500 microliters of FLAG wash buffer. After the third wash, transfer the protein bead solution to a new 1.5 milliliter microcentrifuge tube to reduce background. Wash a fourth time with FLAG wash buffer.

Remove the supernatant from the final spin and add to the beads 30 microliters of FLAG wash buffer containing 200 nanograms per microliter of FLAG peptide. Vortex at 800 rpm for two hours at four degrees Celsius. After two hours, spin down the suspension at 1, 500 times g for two minutes at four degrees Celsius.

Harvest the supernatant and store at four degrees Celsius as the FLAG IP elution. In this video, the Tandem affinity purification, or TAP method, was applied to the Cyclin-T1 CDK9 complex. STREP tagged Cyclin-T1 and FLAG tagged CKD9 were overexpressed in HEK293 T cells.

A reciprocal experiment was also performed with STREP tagged CDK9 and FLAG tagged Cyclin-T1. Western blot analysis shows that FLAG tagged CKD9 coeluted with STREP tagged Cyclin-T1 from the STREP beads, but not in the negative control, AP lacking STREP tagged Cyclin-T1. Both proteins were detected in the FLAG elution, which further confirmed the complex formation.

The reciprocal TAP results show that FLAG tagged Cyclin-T1 coeluted with STREP tagged CDK9, further validating that the Cyclin-T1 CDK9 interaction is independent of the epitopes utilized. The results of the TAP and reciprocal TAP were also analyzed by silver staining. The asterisks indicate coeluted impurities.

FLAG tagged CKD9 coeluted with STREP tagged Cyclin-T1 off of the STREP beads, but not from the control AP.In the subsequent FLAG elution, STREP tagged Cyclin-T1 coeluted with FLAG tagged CDK9 off of the FLAG beads. Similarly, FLAG tagged Cyclin-T1 coeluted with STREP tagged CDK9 off of the STREP beads, but not from the control AP.And the protein-protein complex was efficiently recovered after the second FLAG IP step. Once mastered, this technique can be done in two days, starting when cells are ready to be collected if it is performed properly.

While attempting this procedure it is important to remember to increase the number of cell plates per experiment accordingly to ensure that enough protein can be recovered and detected. For example, identifying the novel regulatory subunits and/or studying the post-translational modifications on complex subunits. After its development, this technique paved the way for researchers studying protein-protein interactions to explore novel interactors and post-translational modifications in eukaryotic cells.

After watching this video, you should have a good understanding of how to isolate and characterize proteins using STREP and FLAG tags.