The radioactive pulse chase using 35 S-labeled methionine and cysteine is still the only biochemical methods to investigate protein biosynthesis with time in live cells. Protein biosynthesis covers both translation, folding and assembly, and trafficking and degradation. Combining the radioactive pulse chase with analysis of co-and post-translation protein modifications, such as disulfide bond formation and acetylation provides a sensitive and quantitative assay to investigate the protein's faith with time.
Good focus and experimental handling is required for this procedure. Good preparation is crucial, like buffers, your radioactive workspace, and a clear schedule. This video provides a clear over the shoulder view of the radioactive pulse chase protocol.
Demonstrating the procedure will be Hui Ying Yeoh, a pHd candidate from our laboratory. To begin this procedure, seed transfect and culture cells as outlined in the text protocol. Before the pulse chase, inspect your cells through the microscope.
Next, wash the dishes with two milliliters of wash buffer. And two milliliters of starvation medium to the cells and place the dishes in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 15 minutes. Make sure that your pulse chase set up is in order.
Transfer the dishes to the racks in the water bath, pre-warmed to 37 degrees Celsius. Make sure the dishes are in contact with water, but do not float. Then, start a timer.
At 40 seconds, aspirate the starvation medium. Using a micro pipet, draw up 600 microliters of pulse solution. At exactly one minute, gently add the pulse solution to the center of the dish.
Repeat this process of removing the starvation medium and adding the pulse solution for the remaining dishes at one minute intervals. At exactly 11 minutes and for all following dishes, according to the pulse chase scheme, except for the zero minute chase sample, add two milliliters of chase medium directly to the dish. Aspirate the chase medium and replace it with two milliliters of fresh chase medium.
Repeat this process for all remaining dishes at one minute intervals. When the timer reads exactly 16 minutes, add two militers of chase medium directly to the zero minute chase dish, on top of the pulse medium, to stop labeling. Immediately aspirate the medium.
Transfer the dish to a cooled aluminum plate and add two militers of ice cold stop buffer. Transfer all other dishes to an incubator at 37 degrees Celsius. Transfer each chase dish back from the incubator to the water bath two minutes before the end of each chase time.
At the exact chase time for each dish, aspirate the chase medium and transfer the dish to a cooled aluminum plate. Add two milliliters of ice cold stop buffer. Leave all dishes on ice and stop buffer until there is time to lyse the cells.
Then aspirate the stop solution and wash all dishes. Three at the same time with two milliliters of ice cold stop solution. Aspirate the stop solution completely and add 600 microliters of lysis buffer to two dishes at a time.
Using a cell scraper, scrape the surface of the dish thoroughly, but gently to mix the lysate. Transfer lysate from each dish to a fresh 1.5 milliliter micro centrifuge tube. Centrifuge at 15, 000 to 20, 000 G at four degree Celsius for 10 minutes to pellet the nuclei.
For the pulse chase on suspension cells collection five million cells per time point in one 50 milliliter tube. Perform the starvation procedure as described in the text. After the starvation procedure in the water bath for 15 minutes, start the timer.
At exactly one minute, add 275 microcuries of undiluted label directly to the tube containing cells and swirl to mix. At exactly 11 minutes, add four milliliters of chase media to stop the labeling. And immediately transfer one milliliter to a 15 milliliter tube on ice that contains nine milliliters of ice cold stop solution to stop labeling.
Repeat this process of transferring one milliliter of chase to a tube on ice containing nine milliliters of stop solution for each successive chase time point. After the immunoprecipitation, aspirate the last wash completely. Re-suspend the beads in 20 microliters of TE buffer and vortex.
Add 20 microliters of two X sample buffer without the reducing agent. Vortex the samples and then heat them at 95 degrees Celsius for five minutes. Vortex the samples again.
Centrifuge at 12, 000 times G at room temperature for one minute. Transfer 19 microliters of the non-reduced supernatant to a fresh microcentrifuge tube that contains one microliter of 500 millimolar DDT. If needed, centrifuge the sample quickly so that all liquids are at the bottom.
And heat at 95 degrees Celsius for five minutes. Let the reduced sample cool down and then vortex. Centrifuge both the reduced and non-reduced samples at 12, 000 times G at room temperature for one minute, and then add 1.1 microliters of one molar NEM to all the samples.
In this study, a radioactive pulse chase approach is used to analyze protein folding and transport in intact cells. The folding and secretion of HIV one GP120 from an adherent pulse chase is shown here. The non-reducing gel shows the oxidative folding of GP120.
Immediately after the pulse labeling, it appears as a diffuse band higher in the gel. As the chase progresses, the band migrates down the gel through even more diffuse folding intermediates until it accumulates in the tight band that represents natively folded at GP120. This occurs as the formation of disulfide bonds increases the compactness of the protein, causing it to migrate faster than the fully reduced protein.
On the reducing gel, the disulfide bonds and all molecules have been reduced and do not affect mobiilty. As such, differences in mobility are only the result of changes in molecular mass. The shift over time from the reduced signal peptide uncleaved form to the reduced signal peptide cleaved form represents the post-translational signal peptide cleavage of GP120.
The mobility increases due to the loss of the signal peptide which increases during the chase as more proteins attain the native fold and lose their signal peptide. On both the non-reducing and reducing gel, the signal begins to decrease from approximately one hour onward due to the secretion of GP120. This can be monitored by analyzing the media.
This method is applicable to any cell line, including organoid cell models. But the success greatly depends on the expression level of the protein of interest. Before beginning this procedure, mark your dishes and keep this order throughout the experiment.
Make sure everything is prepared before adding starvation medium. This is the start of the experiment. And your lab time is your friend, but can be your biggest foe when you're not prepared.
Bill's chase can be combined with limited proteolysis to investigate the full estate of proteins with time. Also different elected freezers methods will allow analysis of rinse or rigormorization or charge differences. To protect the researcher and apparatus, all local radiation safety rules must be obeyed.
And protective measures should be taken according to protocol. Note that your acrylamide, used for your STH page is neurotoxic.
