A common technique to find out a protein's function is to delete it and see what happens. But you can't do that with an essential protein. It's essential, the cell dies.
So you need a way to remove that protein and catch a glimpse of what happens just before cell death. Protein depletion should be fast, so you get no secondary effects. It should be specific, so that only that protein is removed and only depleted when you want it to be.
And it should not affect the cell in any other way other than removing that protein. After preparing the strain, use an overnight culture to determine what dilution is needed. Use this information to set up a sufficient new culture with an OD600 between 0.1 and 0.2, and grow it at 30 degrees Celsius.
In a fume hood, add a methanol equivalent to 30 to 50%of the intended sample volume to a 15-milliliter tube. Close the tubes tightly, label them, and place them on dry ice to chill. Next, label 1.5-milliliter tubes for long-term storage of the samples and place on ice to cool.
Cool at least one milliliter of water per sample on ice. Once the target optical density for the start of the pre-incubation has been reached, transfer a sample into the pre-prepared tube containing cold methanol. Invert the tube briefly and place it back on dry ice.
This is the uninduced control sample. Immediately, add the beta-estradiol such that the final concentration is 10-micromolar. Swirl vigorously to mix.
Continue to grow the culture as before, incubating with beta-estradiol for the optimal time as outlined in the text protocol. The beta-estradiol incubation time is the most important step, and must be optimized for every protein. Too short and the depletion is incomplete and slow.
Too long and the protein levels will fall even before you've added the auxin. Pipette up 0.5 microliters of IAA per milliliter of culture to add later. Collect a sample from the undepleted culture as previously described.
Immediately add the IAA to a final concentration of 750-micromolar and swirl vigorously to mix. Start a timer as soon as possible after mixing. Collect samples according to the experimental design.
Then, place the samples on ice. Ensure that none of the samples have frozen. If any have, gently warm them in the hand while constantly inverting.
Once all of the samples are collected and not frozen, centrifuge them at 3, 500 times G and four degrees Celsius for two minutes. Pour off the methanol and medium mix and place the samples back on ice. Then, resuspend the cell pellet in one-milliliter of water and transfer this suspension to a labeled tube on ice.
Centrifuge briefly at a speed over 15, 000 times G to repellet the cells. After this, place the tubes back on ice and aspirate the liquid. In this study, degradation is tuned to achieve specific and efficient protein depletion without otherwise affecting the metabolism of the yeast cell.
The low-abundance spliceosomal Prp2 and Prp22 proteins are both depleted to less than 20%after 20 minutes of pre-incubation with beta-estradiol, followed by 15 minutes with auxin. Longer pre-incubation times lead to faster depletion, but also show undesirable protein depletion before auxin addition. In comparison, the more abundant Dcp1 is only depleted to approximately 30%with the same treatment.
However, 60 minutes of pre-incubation time results in depletion to 13%with the same auxin treatment at the cost of depletion before the auxin is added. Optimize the pre-incubation time, and don't lose track of time in between your time points. What to do next depends on your question.
We've found that 10-mils sample of culture is enough for protein, DNA, or RNA analysis. The sampling procedure is also easily adaptable for ChIP. Now there is a rapid and specific way to deplete proteins.
So the function even of essential proteins can be found. The protocol is fairly safe. Methanol is the only dangerous chemical.
Take care while dispensing it. Do it in a fume hood, and wear two pairs of gloves, as methanol can get through most nitrile gloves.
