SDS-PAGE Sample Preparation and Assay

This page is part of the SDS-PAGE lab, which includes these pages:

The whole experiment will be spread over three lab days. You will also use these methods as part of the pGLO lab.

This page tells you how to:

  • Prepare a protein lysate starting from a liquid bacterial culture.
  • Perform a protein assay to measure the concentration of protein in your lysate.

The next steps after this are to:

Prepare a protein lysate from a bacterial culture

Some of these steps may already be done for you; your instructor will tell you where to start.

The bacterial samples are cultured in glass culture tubes containing a nutrient broth. The cultures should be cloudy when you receive them, perhaps with a visible mass of cells at the bottom of the tube. The proteins that you want are inside those cells. Before you can see the proteins on a gel, you need to burst open, or lyse the cells. When the cells undergo lysis, the material released from the cytoplasm is called a lysate. The lysate will contain most of the cells’ proteins, along with RNA and other components.

  1. From each culture, pipet 2 ml into a 2-ml microcentrifuge tube. (If you run short, add extra broth so the tubes are balanced.) Use the stuff on the bottom of the tube, because that’s where all the cells are.
  2. Centrifuge for 5 minutes at high speed. Be sure to balance the centrifuge.
  3. After the spin, you should see solid bacterial pellets in all four tubes. At this point, the cell pellet contains the protein you want. Pipet off the liquid supernatants into an empty glass culture tube for disposal.
  4. Add 250 µl of TE buffer to each bacterial pellet. Resuspend the bacterial pellets thoroughly by gently pipetting up and down.
  5. Add 50 µl of lysozyme to each resuspended bacterial pellet and vortex or flick the tube to mix. Let sit at room temperature for 5 minutes. The lysozyme will start digesting the bacterial cell wall, weakening the cells so they can be lysed by freezing.
  6. Put the tubes in the –80° freezer until they’re frozen solid (about 10 minutes). Freezing will complete the lysis of the bacteria, because the ice crystals break the cells open. It’s essential that your samples freeze completely.
  7. Remove your tubes from the freezer and thaw them in your hand. Flick the tubes so they are mixed well. Spin the tubes in the centrifuge for 10 minutes at high speed. This will pellet the insoluble bacterial debris.
  8. After the 10 minute centrifugation, remove your lysate tubes from the centrifuge. The insoluble bacterial debris should be visible as a pellet at the bottom of the tube. The supernatant contains all the soluble materials from the lysed cells – including proteins, and many other kinds of molecules.
  9. Each lysate tube should contain about 300 μl of liquid. Carefully pipet 250 µl of this supernatant into a new tube. Do not disturb the pellets at this point; it’s important to avoid getting any of the pellet material into your new lysate tube. After the spin, the supernatant is called a cleared lysate. You’ll use your cleared lysates for two different things: some for the protein assay, and some for the SDS-PAGE gel.
  10. Dispose of the tubes containing the pellets in the biohazard trash. Label your lysate tubes carefully with your group name, the date, and the name of the bacterial sample. At this point, your lysates are ready to use, or you can store your lysates in the freezer until the next lab. The instructor will show you where to put them.

Perform a protein assay

Background

Electrophoresis works best when you load an appropriate amount of sample on the gel. If you load too much protein, the bands will be smeared; if you load too little, you may not see your bands at all. In order to get the protein amount right, you’ll use a fluorescent protein assay to measure, or quantitate, your protein concentration before loading your gel. The

This protein assay uses a device called the Qubit fluorometer, from Invitrogen Corporation. In general, fluorometers work by illuminating a sample with short-wavelength light and then measuring the amount of longer-wavelength light given off as fluorescence. You’ll use the Qubit fluorometer with the Quant-iT protein assay kit (also from Invitrogen). You’ll mix your protein sample with a special reagent that becomes fluorescent only when it is bound to protein. Next, you’ll place your sample in the fluorometer. If there is no protein in your sample, there will be very little fluorescence, and the fluorometer will give a low reading. The higher the concentration of protein in your sample, the higher the reading will be.

The fluorometric protein assay has two important advantages over some other assay methods. First, it’s very specific. Since the fluorescent reagent in the working solution binds only to protein, you won’t end up accidentally measuring some other kinds of molecules instead of protein. Second, it’s very sensitive, so you can use a small part of your sample for the assay and leave the rest for SDS-PAGE or some other procedure.

The fluorometer can also be used to assay DNA concentration, simply by using a different fluorescent reagent that binds to DNA instead of protein. You’ll do that  in a later lab.

This procedure is simple. You mix the protein samples with the working solution, calibrate the fluorometer, and measure the protein concentration of your samples. However, it’s also very easy to mess up this procedure. If your pipetting is not accurate, the assay won’t work. It's common for 6B students to mess it up on the first day of lab!

One group can prepare the working solution and mix the protein standards with the working solution. If you aren't doing these steps, you should wait until the working solution is ready, then start at Assay your samples below.

Prepare the working solution

The working solution is simply a buffer containing the fluorescent reagent that binds to protein. It is used both in calibrating the fluorometer and in performing the assay. It's best to use one batch of working solution for the whole experiment, so one group can do this for the whole class.

How much to make: Each assay uses just under 0.2 ml working solution. There are 7 groups (tables) in the lab, and each group will have 3 different samples, for a total of 21 sample assays. In addition, there are 3 calibration standards, bringing the total amount of working solution to 24 x 0.2 ml, or 4.8 ml. Make 8 ml of working solution to ensure that we have enough.

How to make it: You’ll need to prepare the appropriate amount of working solution by mixing Quant-iT protein buffer (component B in the protein assay kit) and Quant-iT protein reagent (component A in the protein assay kit) as follows:

 Quant-iT protein buffer 995 μl  2.985 ml  3.98 ml  7.96 ml
 Quant-iT protein reagent  5 μl  15 μl  20 μl  40 μl
 Total working solution  1.00 ml  3.00 ml  4.00 ml  8.00 ml

Use the amount for 8 ml; other volumes are included in case you need to make a smaller amount some other time. You can make any amount simply by mixing protein reagent and protein buffer in a 1:200 ratio.

Use a 15-ml Falcon tube for the working solution; the instructor will show you what that is. Add the two ingredients, put on the lid, and gently invert the tube a couple of times to mix it. Label the tube WS and write the time on it, to distinguish it from other batches made the same day. The working solution is now ready to use.

Mix the protein standards with the working solution

Before performing your assay with a sample containing an unknown protein concentration, you’ll need to calibrate the device using three samples with known protein concentration, called protein standards. These standards come with the assay kit, but they need to be mixed with today’s fresh batch of working solution before the calibration can be performed. Since each batch of working solution could be slightly different, it's important to perform a new calibration each time. Calibration gives the device a precise relationship between a specific protein concentration and a level of fluorescence, which is the physical property that the fluorometer actually measures.

For both the calibration and the assay, you need to use special assay tubes, which are thin-walled and transparent so the fluorometer can get an accurate reading. These tubes aren't used for anything else. For the fluorometer, each tube should contain a total volume of 200 μl, obtained by mixing some working solution and either a protein standard or an unknown protein sample.

There are three protein standards, and only one group needs to prepare them; if we're lucky, someone will volunteer. The procedure is as follows:

  1. Label one 500-µl assay tube for each of the three protein standards (1, 2 & 3). Label by writing on the top with a Sharpie, so you don't block the light traveling through the side of tube inside the fluorometer.
  2. Pipet 190 μl working solution into each tube.
  3. Add 10 μl of protein standard #1 to tube 1.
  4. Add 10 μl of protein standard #2 to tube 2.
  5. Add 10 μl of protein standard #3 to tube 3.
  6. Mix all the tubes briefly. These are your calibration tubes. Write down the time you mixed the samples with the working solution, and allow these tubes to incubate at room temperature for 15 minutes before using them in the fluorometer.

Perform the calibration

  1. Plug in the Qubit fluorometer and turn it on by pressing any button. You should see the HOME screen.
  2. On the HOME screen, use the up or down arrows to select the Quant-iT Protein Assay. Press GO.
  3. Insert standard tube 1. Press GO. The reading will take about 5 seconds. Remove the tube.
    Repeat with standard tubes 2 and 3. When all three readings are done, the calibration is complete.

If the calibration fails, it's generally because the three calibration tubes weren't made properly; the fluorometer needs to detect a specific linear increase from each sample to the next. You probably need to make new standards (the working solution is usually not the problem).

Assay your unknown samples

  1. Label one 500-µl assay tube for each of your the three protein samples (the lysates prepared earlier). Label by writing on the top with a Sharpie, so you don't block the light traveling through the side of tube inside the fluorometer.
  2. Pipet 190 μl working solution into each tube.
  3. Add 10 μl of the appropriate protein sample into each tube.
  4. Mix all the tubes briefly. These are your assay tubes. Write down the time you mixed the samples with the working solution, and allow these tubes to incubate at room temperature for 15 minutes before using them in the fluorometer. During this time the protein reagent is chemically reacting with the protein in your sample to produce a fluorescent product. If the reaction is incomplete, your reading will be incorrect.
  5. Check to make sure the fluorometer is set to the protein assay and has been calibrated.
  6. Insert your sample tube and press GO. The fluorometer should display the protein concentration in the assay tube. Write this number down, including the units (e.g., μg/ml). Repeat with your remaining samples.

If the fluorometer reads OUT OF RANGE, read it carefully. The protein concentration is either too high or too low for a good reading. The solution is to prepare a new assay tube, using either a smaller or larger volume of protein sample and mixing with working solution to make a final volume of 200 μl.

Write down your results in your notes, and also record them on this spreadsheet, so we can compare readings for the whole class.

The protein concentration you read on the screen is the concentration in the assay tube inside the fluorometer — the sample that you have diluted with working solution. In order figure out the concentration of protein in your sample tube (undiluted), you may need to do a little math, as described on the calculations for Protein Gel Loading page.

Review

Terms & Concepts

  • Assay
  • Fluorescence
  • Fluorometer
  • Lyse vs. lysis vs. lysate
  • Lysozyme
  • Sample tube vs. assay tube vs. gel-ready tube in the SDS-PAGE lab
  • Protein standards
  • Working solution

Review questions

  1. The fluorometer can indirectly tell you the concentration of protein in a sample, but what physical property of your sample is it directly measuring?
  2. Why would we use the fluorometric assay instead of some other kind of assay?
  3. Why do you need to let your protein samples incubate with the working solution for 15 minutes before performing the assay.
  4. What do you do if the fluorometer reads "out of range"?
  5. This page describes a protein assay. What makes it specific to protein? Sometimes we also do DNA assays, using a similar procedure. What could make that assay specific for DNA?

 

 

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