The overall goal of this procedure is to generate inducible localization and affinity purification, or LAP-tagged stable cell lines, for investigating protein function, spatiotemporal localization, and protein interaction networks. This method can help answer key questions in molecular and cell biology related to protein function, protein cell cycle subzero localization, and protein interactions. The main advantage of this method is that it is amenable to high throughput analysis of groups of proteins, and can be used to dissect the protein components of biological pathways.
To begin this procedure, clone the open reading frame of the gene of interest into the LAP-tagged vector and transfect it into HEK293 cells as described in the text protocol. One day post-transfection, replace the minus Tet DMEM/F12 media with fresh media. Two days post-transfection, split the cells to 25%confluence.
Allow the cells approximately five hours to attach. Then, add hygromycin-containing minus Tet DMEM/F12 media at the predetermined concentration. For HEK293 cells, use 100 micrograms per milliliter hygromycin.
Replace the media as needed until distinct cell foci appear that resemble opaque spots against the transparent plate. Add 20 microliters of trypsin on top of each cell foci and pipe it up and down two times with a 200 microliter pipette tip. Transfer the cells in a 24-well plate and expand the cells by continual growth in hygromycin-containing minus Tet DMEM/F12 media.
Expand the validated LAP-tagged cell line for tandem affinity purification, or TAP, isolation of protein complexes. To do so, continually passage all the HEK293 cells into larger plates and/or roller bottles, and minus Tet DMEM/F12 media at 37 degrees Celsius and five percent carbon dioxide. For Tet/Dox inducible cell lines, induce the cells for 10 to 15 hours by adding a concentration of 0.2 micrograms per milliliter Tet/Dox when the cells reach approximately 70%confluency.
Harvest the cells by agitation or trypsinization. Then, pellet them at 875 times g for five to 10 minutes. To couple the anti-GFP antibody to protein A beads, equilibrate 160 microliters of packed volume protein A beads with PBST in a 1.5 milliliter tube.
Wash the beads three times with 1 milliliter of PBST. Following each wash step throughout this procedure, the beads are centrifuged at 5000 times g for 10 seconds, and the supernatant is removed. After re-suspending the beads in 500 microliters of PBST, add 80 micrograms of affinity purified rapid anti-GFP antibody to each tube, containing 160 microliters of beads.
Mix for one hour at room temperature. Wash the beads two times with one milliliter of PBST, then wash the beads two times with one milliliter of 0.2 molar sodium borate, pH After the final wash, add 900 microliters of the sodium borate buffer, to bring the final volume to one milliliter. Then, add 100 microliters of 220 molar DMP to the bead suspension, for a final concentration of 20 millimolar.
Rotate the tubes gently at room temperature for 30 minutes. After incubation with DMP, wash the beads one time with one milliliter of 0.2 molar ethanolamine, 0.2 molar sodium chloride pH 8.5, to inactivate the residual crosslinker. Then, re-suspend the beads in one milliliter of the same buffer, and rotate for one hour at room temperature.
After pelleting the beads, re-suspend them in an additional 500 microliters of the buffer. The prepared beads are stable for several months at four degrees Celsius. To prepare the cell lysates, re-suspend 500 microliters of packed cell volume into 2.5 milliliters of LAP 300, with 0.5 millimolar DTT and protease inhibitors.
Add 90 microliters of 10%nonyl phenoxypolyethoxylethanol, and mix by inversion. Place the mixture on ice for 10 minutes. Centrifuge at 21, 000 times g for 10 minutes.
Following centrifugation, collect this low-speed supernatant and set aside a 10 microliter sample for gel analysis. After transferring the low-speed supernatant to a TLA 100.3 tube, spin at 100, 000 times g for one hour at four degrees Celsius. Collect this high-speed supernatant in a tube and place on ice, reserving a 10 microliter sample for gel analysis.
For the first affinity capture through binding to anti-GFP beads, pre-elute the antibody-coupled beads by washing them three times with one milliliter of elution buffer to remove the uncoupled antibodies and reduce background. Perform this quickly. Do not leave beads in high salt for a long time.
Then, wash the beads three times with one milliliter of LAP 200 N.Next, mix the high-speed supernatant extract with antibody beads for one hour at four degrees Celsius. After centrifuging the extract at 21, 000 times g for 10 minutes, reserve a 10 microliter sample of the supernatant for gel analysis. Perform three quick washes of the beads with one milliliter of LAP 200 N, containing 0.5 millimolar DTT and protease inhibitors.
Use the same buffer to wash the beads two additional times with a five minute incubation time for each wash. Then, wash the beads quickly two more times with 1 milliliter of LAP 200 N containing 0.5 millimolar DTT and no protease inhibitors, before adding the Tobacco Etch Virus, or TEV, protease. Add 10 micrograms of the TEV protease in one milliliter of LAP 200 N to the beads, and rotate the tubes at four degrees Celsius overnight.
The next day, pellet the beads and transfer the supernatant to a fresh tube. Rinse the beads twice with 160 microliters of LAP 200 N, containing 0.5 millimolar DTT and protease inhibitors to remove any residual protein. For the second affinity capture through binding to S-protein agarose, wash one tube of 80 microliters S-protein agarose slurry three times with one milliliter of LAP 200 N.Add the TEV eluted supernatant to the S-protein beads, and rock them for three hours at four degrees Celsius.
After pelleting the beads, wash them three times with one milliliter of LAP 200 N containing 0.5 millimolar DTT and protease inhibitors. Then, wash the beads two times with one milliliter of LAP 100. Elute the proteins off the S-protein agarose by adding 50 microliters of 4x Laemmli sample buffer and heating at 97 degrees Celsius for 10 minutes.
To identify interacting proteins by mass spectrometry analysis, test the quality of the purification by analyzing the collected samples by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Silver stain the resulting gel. Also, immunoblot the eluates and probe with anti-GFP antibodies to ensure the LAP-tagged purification worked.
To identify stoichiometric and substoichiometric co-purifying species, take the final elution sample and separate it by SDS-PAGE. Stain the gel with a mass spectrometry compatible protein stain. Excise the most prominent bands in the gel and the space in between them to process the bands separately for analysis by mass spectrometry.
Shown here is a representative western blot analysis of the protein samples from non-induced and Dox-induced LAP-Tau HEK293 cells. The cells are probed with anti-Tubulin antibodies to detect the Tubulin loading control, and probed with anti-GFP to detect the LAP-tagged Tau protein. Note that LAP-Tau is only expressed when the cells are induced with Dox.
Mytotic cells expressing LAP-Tau were fixed and co-stained for DNA and Tubulin with anti-Tubulin antibodies, and the sub-cellular localization of LAP-Tau was analyzed by fluorescence microscopy. Note that LAP-Tau localizes to the mitotic spindle and spindle poles during mitosis. Shown here is a representative silver stained gel of the LAP-Tau purification.
The lanes represent the molecular weight, the cleared lysates, and the final eluates. Samples were run on a 4-20%SDS-PAGE gel and the gel was silver stained to visualize the purified proteins. Note that a band corresponding to LAP-Tau is marked with an asterisk, and several other bands corresponding to co-purifying proteins can be seen.
After watching this video, you should have a good understanding of how to generate inducible LAP-tagged stable cell lines, and how to perform biochemical purifications of LAP-tagged proteins for proteomic analysis and identification of protein interaction networks.
