Protein Characterization Using the Multisizer 4 Coulter Counter

Beckman Coulter’s Multisizer 4 offers a user-friendly, technologically-sophisticated system that can resolve most counting and particle sizing challenges. The Multisizer 4 has utilized the Coulter Principle with Smart Technology, ensuring uniformity, consistency of sample analysis and repeatability of results. The cleaning procedures are valuable when characterizing very small particles at low concentrations. This article discusses in details the analysis procedures.

Material Requirements

  • Nalgene 5 mL Sample Vial (Fisher P/N 6250-0005).
  • Stage Adapter (Beckman Coulter P/N A93166).
  • Neutral Detergent Cleaner (Beckman Coulter P/N 8304096).
  • Recommended Apertures: 30 μm, 50 μm, 70 μm, or 100 μm.
  • Internal Aperture Brush (Beckman Coulter P/N 54060001).
  • External Aperture Brush (Beckman Coulter P/N 5406011).
  • Accuvette ST (Beckman Coulter P/N A35473).
  • 200-mL Enhanced Performance Beaker (Beckman Coulter P/N A35596).
  • Isopropyl Alcohol (optional).
  • Additional Electrolyte Jar for 2% Soap Solution (Beckman Coulter P/N A36403) (optional).
  • Syringe filters.
    • Pall Acrodisc 25 mm PES Filters.
    • 0.45 μm pore (Beckman Coulter P/N 4614).
    • 0.20 μm pore (Beckman Coulter P/N 4612).

Procedures

It is important to note that all buffers must be highly filtered to ensure precise counting. It is recommended that buffers are filtered through the 0.45 μm filter, followed by the 0.2 μm filter connected in serial. For convenience, continuous filtration with PES filters is recommended.

Instrument Start-up

In the case of study analyzed in this article, the Multisizer 4 has been idle for more than 72 h; repeated flushing with a 2% solution of neutral detergent is recommended. Optionally, isopropyl alcohol (IPA) can be used. Any salt crystals, dirt or other contaminants are removed, which may have been formed in the system while it was idle.

Start-up Procedure

The start-up procedure of the Multisizer 4 comprises the following steps:

  1. Choose “Run”, then “Drain System” in the menu bar of the Multisizer 4 software. Follow the on-screen instructions.
  2. On draining of the system, fill the electrolyte bottle with cleaning solution (either 2% soap or IPA).
  3. Replace the electrolyte bottle in the Multisizer 4.
  4. Select “Run”, then “Fill System” in the Menu bar.
  5. After filling of the system, repeat steps 1-4. Repeat the entire cycle three times.
  6. After the final drain, fill the system with highly-filtered Isoton II which should come from a continuous filtration system.

It is important to note that the internal waste tank may become full during this process. In that case, “Empty Waste Tank” must be selected when prompted. This will pump the contents of the internal waste tank to the external waste jar.

Running Samples

Based on the type of sample system being run as well as the guidelines presented in Table 1, an aperture needs to be selected with which to work. “Run” has to be selected from the menu bar, then “Change Aperture Tube Wizard” must be selected. The on-screen instructions need to be followed.

Table 1. Guidelines for buffer strength and lowest recommended size range.

Aperture Lowest Recommended Conductivity (μS/cm) Approximate NaCl Concentrationin DI Water Recommended Lower Threshold at this Conductivity
20* 15,000 154 mM 0.4 μm
30* 15,000 154 mM 0.6 μm
50 5,000 40 mM 1.1 μm
70 3,750 30 mM 1.4 μm
100 2,750 20 mM 2.3 μm

* Not recommended for low-conductivity buffers.

The steps to be followed are listed below:

  1. Install the stage adapter. The Sample Stage as it will appear at the onset of experiments is shown in Figure 1.

    The mini-cuvette adapter allows users to operate the Multisizer 4 with as little as 4 mL of material.

    Figure 1. The mini-cuvette adapter allows users to operate the Multisizer 4 with as little as 4 mL of material. Image credit: Beckman Coulter

  2. Fill a 5 mL Nalgene cup with the buffer of interest. It is important to note that for accurate results, this buffer must match the buffer in the electrolyte jar.
  3. Test the noise level in the buffer of interest. From the menu bar, choose “Run”/“Troubleshooting”/“Check the noise level”. A pop-up box will appear. Choose “Measure Noise Level” as shown in Figures 2A and 2B.

    Screenshot of the “Check the noise level” command under Run/Troubleshooting Menu.

    Figure 2A. Screenshot of the “Check the noise level” command under Run/Troubleshooting Menu. Image credit: Beckman Coulter

    Screenshot of the “Check Noise Level”command. After selecting “Measure Noise Level”, the results should match or exceed the recommendations in Table 1.

    Figure 2B. Screenshot of the “Check Noise Level”command. After selecting “Measure Noise Level”, the results should match or exceed the recommendations in Table 1. Image credit: Beckman Coulter

  4. Check whether the standard operating mode (SOM) settings are configured as indicated. Choose “Edit SOM” to bring up the SOM menu. See Figures 3A and 3B.

    Screenshot of the “SOM” Menu. Select “Edit SOM” to open SOM menu.

    Figure 3A. Screenshot of the “SOM” Menu. Select “Edit SOM” to open SOM menu. Image credit: Beckman Coulter

    Screenshot of the “Edit the SOM” Menu. Use the tabs in the SOM menu to navigate. Ensure all the settings match those described in the text.

    Figure 3B. Screenshot of the “Edit the SOM” Menu. Use the tabs in the SOM menu to navigate. Ensure all the settings match those described in the text. Image credit: Beckman Coulter

    1. Under the “Control Mode” tab - Verify whether the control mode is set to volumetric. The maximum recommended analytic volume is 100 μl per run. Select “Apply”.
    2. Under the “Run Settings” tab - Ensure “Include Pulse Data” is checked. Number of Runs must be 3. Select “Flush Aperture tube after each run” and “Flush Aperture tube before first run”. Select “Apply”.
    3. Under the “Stirrer” tab - Check whether “Accuvette ST” is selected under “Sample Beaker”. Click “Apply”.
    4. Under the “Current and Gain” tab - Verify the sizing threshold is at or below the lowest recommended size in Table 1.
    5. Under the “Pulse to Size Settings” tab - Verify that the setting is at or above the lowest recommended size listed in Table 1. Select “SOP” in the menu bar, then “Edit the SOM.” Choose the “Pulse to Size Settings” tab. Check the value in the box next to the word “from”. Select “Apply”.
    6. Under the “Concentration” tab - Enter the dilution information if applicable.
    7. Under the “Blockage” tab - Select “Blockage Detection”. Select “Defaults” then “OK”. Select “Blockage Monitor…”, “Defaults” and then select “OK”.
  5. Once the SOM protocol is done, perform a buffer only sample by selecting “Start”. A buffer only sample must read less than 100 total particles in a 100 μl sample. In order to validate this, ensure that “Number” under the graph drop down menu in the data window has been selected. Then, check the particle count in the statistics box. See Figure 4 for reference.

    Screenshot of the buffer only sample run. The total number of particles should be less than 100 for a run of a 100 µl blank. “Number” should be less than 100 for analysis of 100 µl from a blank

    Figure 4. Screenshot of the buffer only sample run. The total number of particles should be less than 100 for a run of a 100 μl blank. “Number” should be less than 100 for analysis of 100 μl from a blank. Image credit: Beckman Coulter

  6. Once a buffer only run is complete, the instrument is ready to run samples. Remove the blank, load the sample, and select “Start”.
  7. In case a block occurs, follow step 8 “After run is complete”.
  8. After run is complete, lower the stage. Fill a beaker or Accuvette with a 2% soap solution and submerge the aperture can hold beaker in hand and leave the door open. Select “Unblock” in the Multisizer 4 software. This will repeatedly flush the soap solution through the aperture and remove any proteins. This is necessary as proteins tend to adhere to all surfaces. After the unblock procedure is complete, load the blank sample, close the door, and select “Preview”. The concentration meter should be steady at 0.0%. It may take a few seconds before it stabilizes. This is the time necessary to flush away any remaining sample as shown in Figures 5A and 5B.

    With the door open, hold a beaker filled with 2% soap solution around the aperture.

    Figure 5A. With the door open, hold a beaker filled with 2% soap solution around the aperture. Image credit: Beckman Coulter

    While holding the beaker with soap solution, select “Unblock” button in the software.

    Figure 5B. While holding the beaker with soap solution, select “Unblock” button in the software. Image credit: Beckman Coulter

  9. Repeat steps 6-8 until all samples are completed.

Instrument Shut Down Overnight

  1. Fill a 200 mL beaker with 2% soap solution. Flip the stage over so that the beaker can be accommodated. Place the beaker on the stage, but do not raise it.
  2. In the Multisizer 4 software, select “SOP” in the menu bar. Select “Edit the SOM”. Under “Sample Beaker”, select “200 mL ST”. Ensure that the “Use Stirrer” box is selected and the stirrer speed is 20. Select “Apply”. The stirrer must move into position on the Multisizer 4.
  3. The stage is raised with the 200 mL beaker. The stirrer must begin moving once submerged. The system can be left overnight with this set-up. The next day, the “Running Samples” section of this procedure need to be followed.

This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc. - Particle Characterization.

For more information on this source, please visit Beckman Coulter, Inc. - Particle Size Characterization.

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