Study of Protein-protein Interactions in Autophagy Research

My name is Devrim Gozuacik. We are working in Sabanci University, Istanbul, Turkey, on regulation of autophagy in mammals. We are focusing on different proteins, pathways, RNAs regulating autophagy.

There are several techniques to study autophagy. We are using molecular biology, biochemistry, and cell biology techniques to analyze different pathways that we are discovering. We are using immunoprecipitation, immunofluorescence techniques, confocal microscopy, and various other techniques to analyze autophagy pathways.

Details of different techniques and pitfalls and troubleshooting will be explained by my PhD student, Secil Erbil. The overall goal of immunofluorescence and immunoprecipitation procedures is to monitor endogenous protein-protein interactions in a cell. This method can help you to answer the key questions about protein-protein interactions and the cellular localization of them.

The main advantage of these techniques is the opportunity to quantify the interaction intensity during desired treatments and conditions in comparison to control. To begin, detach the cells with trypsin. Then count the cells using a hemocytometer, and calculate the cell concentration.

Take packed and autoclaved cover slides, and place them in a 10-centimeter Petri dish. If you are working with an easily-detaching cell line, such as HEK293T, like in this experiment, coat the cover slides with sterile-filtered 0.01%poly-L-lysine solution. After 10 minutes of incubation, remove the solution.

Wait until all the poly-L-lysine evaporates and the cover slides get completely dry. Then wash them with sterile PBS. Put one milliliter of media into 12-well plate, and place the cover slides into each well.

Seed 20, 000 cells per well. It is important to shake the plate during and after the inoculation in order to have a homogeneous distribution of the cells on cover slides. Treat your cells with desired drug and its control in a way that the total incubation time of the cells do not exceed 48 hours.

At the end of 48 hours, remove the media on cover slides and wash the cells once with PBS. Then, under a fume hood, remove PBS and fix the cells by incubating them with 4%PFA for 20 minutes at room temperature. Since PFA is sensitive to light, cover the plate with aluminum foil during the incubation time.

Then remove PFA, and wash the samples three times with PBS. In this step, it’s important to wash the samples one by one to not dry them. Then label a six-well plate according to your treatment conditions, and cover each well with Parafilm.

In this step, pay attention to have a smooth bottom after Parafilm covering. Then place the cover slides in six-well plate, and put 100 microliter of BSA-saponin mixture on each cover slide, and incubate them 30 minutes on ice for blocking. Then remove the blocking solution, and wash the samples once with PBS.

Prepare one to 100 primary antibody in BSA-saponin mixture, and put 100 microliters of this solution on each cover slide. Incubate one hour at room temperature. During incubation, it’s better to shake the plate in order to have an equal distribution of the antibody on cover slides.

Then remove primary antibody, and wash the samples three times with PBS. Prepare one to 500 Alexa Fluor secondary antibody in BSA-saponin mixture, and put 100 microliters of this solution on each cover slide. Since Alexa Fluor is sensitive to light, it’s important to keep the samples in dark after this step.

Therefore, cover the six-well plate with aluminum foil, and incubate it one hour at room temperature on the shaker. Then remove the secondary antibody, and wash the samples three times with PBS. Label the slides with your treatment names, and clean them with ethanol using Kimwipes.

Then put 10 microliters of mounting solution in the middle of each slide. With a pincette, place each cover slide on the slides. Then take the excess amount of mounting solution gently by using Kimwipe.

First, stabilize each cover slide with four drops of nail polish. Then seal the cover slide completely. Analyze the sample using a confocal microscope.

Here you see an example of colocalization result obtained by using this protocol retrieved from our recent paper. In this video, endogenous RACK1 protein was stained with green, while endogenous LC3 was stained with red. The yellow dots that occur in the Merge pictures are the places where green and red signals overlap.

In other words, yellow dots represent the colocalization of these two proteins. Immunoprecipitation is another commonly used method for studying protein-protein interactions. To begin, seed four million HEK293T cells in 15-centimeter Petri dishes for each condition.

Treat the cells with desired drugs in a way that the total incubation time of the cells do not exceed 48 hours. After 48 hours of incubation, harvest the cells with ice-cold PBS. HEK293T cells detach from the plate easily.

Therefore, washing the plate with PBS by pipetting is enough to detach and harvest all the cells. Collect your cells in Eppendorf Tubes, and centrifuge them 15 minutes at maximum speed at four degrees. Then discard the supernatant, and wash the cell pellet with one milliliter of PBS by resuspending it by pipetting.

Then centrifuge again, and discard the supernatant. Then resuspend the cell pellet in RIPA buffer containing protease inhibitors. Vortex the suspension for 15 seconds, and keep the Eppendorf Tube on ice for five minutes.

Repeat vortexing five times once in five minutes. Then centrifuge the sample at the highest speed for 15 minutes at four degrees. Then take the supernatant and centrifuge again to completely get rid of the cell debris.

Dilute protein lysates one to 50, and prepare a 96-well plate with the blank, standards, and the diluted samples. Mix them with Bradford solution in one to 20 ratio, wait 15 minutes in dark, and measure the optical density at 595 millimeter, and calculate the protein concentration using absorbance values for each sample. Then prepare the beads for antibody coupling.

Cut the edge of a 200-microliter tip using scissors, and take 25 microliters from bead slurry for each condition. Wash the beads once with PBS, once with RIPA buffer, and once with RIPA buffer containing protease inhibitors. After the last wash, resuspend the beads in 300 microliters of RIPA buffer containing protease inhibitors and add 1.5 microgram of antibody which is specific to the protein you want to pull down.

Incubate antibody and bead mixture overnight at four degrees on rotator. After the incubation, wash the beads with RIPA buffer containing protease inhibitors and load two milligrams of total cell lysate from each condition. Fill the total volume up to 300 microliters with RIPA buffer containing protease inhibitors, and incubate them overnight on rotator at four degrees.

Then wash the beads three times with RIPA buffer containing protease inhibitors. After the last wash, by using a smaller pipette, try to take all the supernatant without touching the beads. Then add 10 microliters of 3X loading dye.

For controlling endogenous expression of proteins that will be checked in co-immunoprecipitation experiment, prepare input samples by using at least 100 micrograms of protein lysate. Add appropriate volume of 3X loading dye on each sample. To denature IP and input control samples, boil them at 95 degrees for 10 minutes.

Then spin down and load the samples into SDS gel in order to separate the proteins according to their size by performing western blot. Run the protein samples through SDS gel, and then transfer the proteins to nitrocellulose membranes. After the wet transfer is done, blot the nitrocellulose membrane while incubating it with 5%nonfat milk solution one hour at room temperature on the shaker.

Then wash the membrane three times in PBS-Torin solution five minutes on the shaker at room temperature. Incubate the membrane in the primary antibody which is specific to the protein you want to detect for one hour at room temperature. You may prefer to prepare primary antibodies in red solution to reuse them without losing their quality.

After primary antibody incubation, repeat PBS-Torin washing steps three times. Then prepare HRP-conjugated secondary antibody solution in 5%nonfat milk, and incubate with the membrane for one hour at room temperature on the shaker. After the incubation, again, repeat the washing steps.

Then prepare homemade ECL solution, and place the membrane into a cassette. Then continue developing your fixing steps, as described in the text in a complete diagram. After fixing and drying the film, mark according to the edges of the membrane and protein letter to be able to predict the specific band.

Repeat all these antibody incubation steps followed by darkroom procedures for all proteins that you want to check for interaction. Then analyze the results. Here you see an endogenous immunoprecipitation result retrieved from our recent paper.

In this video, endogenous ATG5 protein was precipitated, and the levels of endogenous RACK1 co-immunoprecipitation was checked under different conditions. While attempting these procedures, it’s important to remember to have as much biological repeats as possible with positive and negative controls in order to collect the quantified interaction levels upon different treatments. The overall picture that all biological replicates give is defined as the pattern of interaction under different conditions.

After watching this video, you should have a good understanding of how to search dynamics of protein-protein interactions.