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Overview of Protein Turnover using 35S: how to prevent trashy data and uh-oh moments

Posted in: Protein Expression and Analysis
Overview of Protein Turnover using 35S: how to prevent trashy data and uh-oh moments

The half-life of a protein is an important factor in many molecular biology studies. If your thesis has anything to do with proteins then your graduate advisory committee will ask about half-life, so start planning your 35S experiment today. To help you here is my overview of the major steps of 35S labeling complete with tips, common mistakes and how to stop other people from messing you up (because no one likes working with radioactivity)!

Step 1: Plan your experiment

I know you are saying “duh” but with radioactivity this is especially true. There is nothing worse than hopelessly wandering around the halls with radioactivity in one hand and a Geiger counter in the other. Before performing a radioactive study you should do a cold “walk through” and consider these factors:

Have you talked to your institution?

Before you do anything you need to contact you institution’s radioactivity officer/department. Make sure you receive the proper training and follow your institution’s guidelines whenever working with radioactivity.

How long do you expect the half-life to be?

This will influence how many time points you need and how long your experiment will last. This is important info to know if you are using shared equipment!

How much radioactivity will I need?

Your dish size, volume of media, replicate number, treatment conditions and number of plates will influence the amount of radioactivity you need to order. Also when ordering radioactivity, make sure you know how long it takes to arrive thought the safety channels at your university.

Where are you going to incubate your cells?

Unfortunately, 35S is votile and will leech out of your experiment into the incubator and the centrifuge. Ideally you will have a breathable, activated charcoal lined chamber to absorb the votile 35S during the pulse inside the incubator. If not, ask environmental safety or other labs for help. Activated charcoal works wonders to absorb 35S from the air in your incubator.

Is anyone else going to be using radioactivity that day?

You get to the lab at six in the morning and three people are using all the Geiger counters. Don’t let this happen to you.

Step 2: Plate your cells

It’s important to plate your cells correctly and consistently, so don’t skip counting and even plating of your cells. And because labeling requires numerous media changes and washes, you may need to coat your plates with poly-D-lysine to help them stick tightly. Also remember that this will be a time course and you will likely need to take one time point at a time. Therefore you may want to opt for individual plates (like 35mm dishes) rather than multi-well dishes (like six wells).

Step 3: Experimental treatments

If you need to compare half-lives under two different conditions, make sure you plan out how and when to add your drugs and transfections, and how this will affect the amount of radioactivity and replicates you will need. You made need a piece of paper, a pen, and time to do this right!

Step 4: Deplete non-radioactive amino acids

You need to purchase cysteine-, methionine- (or whatever amino acid you are labeling)-free media and perform a depletion of these amino acids before adding your radioactive analog. You will also need to dialyze some serum to remove the amino acids and use this during the depletion and the pulse. This step is traditionally performed for about 15 minutes but may cause stress to your cells, so don’t go over time and make sure all your solutions are warmed and ready to go for the pulse, washes and chase before you start (don’t forget it takes time to thaw your frozen 35S cys/met).

Step 5: Pulse

This is the most radioactive step and should be performed in a radioactive safe space. The concentration of radioactivity and length of pulse will depend on the expected half-life ­– typically a range of 0.1–0.2 mCi/ml of radioactive amino acid. Try to use the smallest volume you can without compromising the health of your cells and remember to use the dialyzed serum (10%). When in doubt, always go with the larger volume; it’s better to use a little more radioactivity now then having to repeat the experiment.

Step 6: Wash

I consider this an artistic step simply because everyone will do this a bit differently but it is important to do it right. You must work quickly, gently, not spill and keep tabs on your radioactivity. Washing four times in warm PBS or amino acid supplemented media may not be enough to prevent continued labeling past your zero time point. Theoretically, because half-life is an exponential decay, it shouldn’t matter when zero begins; therefore stressing out about washing length or waiting 5–15 minutes into the chase before you collect the zero time point shouldn’t affect your calculation and may make a better figure.

Step 6: Chase

Your chase media should be double the volume of the pulse media and should contain an excess of the cold (not radioactive) amino acids used in labeling (around 15 mg/L). When you collect your time points be very consistent: wash your cells with cold PBS the same number of times, lyse in the same amount of media, etc. Always assume your protein is super labile, keep your samples cold and add those proteinase inhibitors to your lysis buffer.

Expert Tip: Remember that your cold PBS becomes hot after use. So make sure that you dispose of it as your institution requires.

Step 7: Protein isolation and gel electrophoresis

This experiment is quantitative, so everything should be done precisely or else you will be repeating this experiment more than you, and the environment, want. Assuming you are doing a pull-down assay, keep extra gloves and that Geiger counter close because things will get messy. Distractions are your enemy here; tell that annoying person to stop talking to you while you work on this experiment, even if it’s your PI. Total incorporation of radioactivity using a TCA precipitation is a complete waste of time in my book. You can tell after the wash if incorporation worked using the Geiger counter, and again, this is a decay so your data shouldn’t change depending on how well you labeled.

Step 8: Fix and dehydrate your gel

Although conceptually simple, this step requires great care to prevent tearing and cracking. For starters, double-check that you will have access to the gel dryer when you need it; a BIG SIGN never hurts. Fix your gel for about 30 minutes by emersing it with fixing solution (10% acetic acid, 20% methanol). However, if you have a thick gel, a high percentage (of acrylamide) gel or are using any commercial gel, you NEED to add either a sprinkling of PEG (polyethylene glycol, literally sprinkle it on during the fix) or 3% glycerol to the fixing solution to prevent cracking. Don’t take your gel off too early or let it get too hot too fast or else it will crack and you will be in salvage mode. Not fun.

Step 9: Expose, develop and measure the half-life

In the days of digital, you probably want to start by using a phosphorimager screen. Make sure you use the one that no one has ruined or else you may end up with streaks or blotchy data. Don’t forget to blank it with a white light and make sure you are using the appropriate type of screen (there are different types). It’s always a good idea to do both imager and film. Film capturing at room temperature is typically okay, but if your signal is very strong or weak you may want to try exposing your film in the –80 to stabilize the film chemistry and to increase linear data acquisition. Plot the normalized densitometry of your samples and see how well they fit to an exponential-decay curve (i.e, examine the R2 value). Hopefully your data will fit well to the curve and you can calculate a half-life.

Step 10: Optimize

If you are like most people, the first try is going to be like the first pancake and will go to the dog (not literally of course). And due to the quantitative nature of these experiments you may need to average replicates for each experiment. Therefore, you may need to do your experiment 3–4 times before you get good statistics and a solid half-life, especially if you have multiple conditions (I have a headache just thinking about it). Here are some other parameters that may need adjusting in order to get meaningful data.

  • Concentration of radioactivity
  • Length of pulse
  • Cell number and confluency
  • Expression of protein of interest (endogenous vs stable vs transient vs time after transfection)
  • How you isolate your protein of interest (the antibody used in the IP, the buffers/protocol)
  • Is your protein degrading after collection?

Good luck!

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