Skip to main content
This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected] Learn more

Coomassie Purity Stain of Recombinant Antibodies


Introduction

This protocol describes how to determine the purity and concentration of recombinant antibodies using ready-to-use bio-safe Coomassie G-250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard antibody of known concentration. After staining with Coomassie blue, protein band intensities are measured using ImageJ software and a standard curve is generated. The ratio of the antibody protein content to the total protein content of the sample is determined.

Sharing speeds science. We believe that sharing the full details of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.


Last Update: March 2, 2022

Workflow Timeline

  • Day 1: Run SDS-PAGE and stain gel
  • Day 1 or later: Image analysis

Equipment

  • Heat block
  • 1–10 µL single channel pipette
  • 2–20 µL single channel pipette
  • 20–200 µL single channel pipette
  • 200–1000 µL single channel pipette
  • Pipette controller
  • Microcentrifuge
  • Electrophoresis chamber
  • Power supply
  • Rocking platform
  • Fume hood
  • Metal spatula
  • Razor blade
  • Plastic tray
  • Gel imaging system
  • ImageJ or similar software

Reagents

  • Purified recombinant antibody, 0.9–1.1 mg/mL
  • SimplyBlue SafeStain, Thermo Fisher, LC6060
  • 4-12% NuPage Novex bis-tris mini gel, 1mm thick, 10-well, Invitrogen NP0321BOX
  • 20X MOPS SDS running buffer, Invitrogen NP0001
  • 4X NuPage sample buffer, Invitrogen NP0007
  • 10X NuPage sample reducing agent (500 mM dithiothreitol, DTT), Invitrogen NP0009
  • PageRuler Plus Prestained protein ladder, Invitrogen 26619
  • Pipette tips, 10 µL, VWR 76322-132
  • Pipette tips, 20 µL, VWR 76322-134
  • Pipette tips, 200 µL, VWR 76322-150
  • Pipette tips, 1000 µL, VWR 76322-154
  • Pipettes, 5 mL, VWR 89130-896
  • Pipettes, 10 mL, VWR 89130-898
  • Pipettes, 25 mL, VWR 89130-900
  • Pipettes, 50 mL, VWR 89130-902
  • Gel loading tips, Corning 4853
  • Microcentrifuge tubes, Neptune 3745.X
  • PBS, 1X pH 7.4, VWR 45000-446
  • 250 mL sterile bottles, Corning 430281
  • Deionized water
  • IgG isotype standard 2.5 mg/mL - can use commercial standard or validate an in-house standard

Before Starting

Warm the hot plate to 100 °C.

Thaw IgG standard and prestained protein ladder on ice.

Reagent Preparation

Prepare IgG Standard

  • Dilute the 2.5 mg/mL IgG isotype standard in PBS as follows:
    • 1.5 mg/mL: 18 µL of 2.5 mg/mL with 12 µL PBS
    • 1.0 mg/mL: 20 µL of 1.5 mg/mL with 10 µL PBS
    • 0.75 mg/mL: 15 µL of 1.0 mg/mL with 5 µL PBS
    • 0.5 mg/mL: 13.3 µL of 0.75 mg/mL with 6.7 µL PBS
    • 0.25 mg/mL: 10 µL of 0.5 mg/mL with 10 µL PBS
    • 0.125 mg/mL: 10 µL of 0.25 mg/mL with 10 µL PBS
  • Transfer 5 µL of each standard to a microfuge tube.
  • Add 8 µL of PBS to a total volume of 13 µL.

Prepare the recombinant antibody

  • Dilute or concentrate purified recombinant antibody to a concentration of 0.9–1.1 mg/mL.
  • Transfer 5 µL of recombinant antibody to a microfuge tube.
  • Add 8 µL of PBS to total 13 µL.

Prepare 1X MOPS buffer

  • Dilute 25 mL of 20X MOPS buffer into 475 mL of deionized water.
  • Gently invert to mix.

Procedure

Section 1: SDS-PAGE

  1. Add 5 µL of 4X sample buffer to each sample.
  2. Add 2 µL 10X reducing agent to each sample.
  3. Spin the sample briefly in the microcentrifuge.
  4. Heat the samples for 10 min at 100 °C in a heat block.
  5. Spin the sample briefly in the microcentrifuge.
  6. Remove the gel from the plastic wrapper and rinse with deionized water.
  7. Gently remove the white sticker on the bottom of the gel.
  8. Place the gel in the electrophoresis chamber and secure it.
    • Note: Check the manufacturer’s instructions if you are unsure of the correct orientation of the gel.
  9. Carefully remove the comb from the gel.
  10. Rinse each well with 200 µL 1X MOPS running buffer.
  11. Fill the chamber with 1X MOPS running buffer.
  12. Make sure that the chamber is sealed.
  13. Load 5 µL of the prestained protein ladder to the appropriate well.

    *Pro-Tip* When possible, skip one lane before loading samples.

  14. Load 20 µL of each recombinant antibody sample to the appropriate well.
  15. *Pro-Tip* Leaving clear lanes between samples will make quantifying the gel easier.

  16. Cover the electrophoresis chamber and attach to a power supply.
  17. Run the gel at 150 V for 1 h.
  18. *Pro-Tip* If the samples are running unevenly and the dye front looks like a “smile”, reduce the voltage.

  19. Turn off the power supply and unplug the electrophoresis chamber.
  20. Remove the gel from the chamber.
  21. Use the metal spatula to gently break the gel cast open.
  22. Use a razor blade to cut the top of the wells and bottom part of the gel where dye is visible.

Section 2: Staining the Gel

  1. Place the gel in a plastic tray with 100 mL of deionized water.
  2. Rinse gel with deionized water for 5 min with gentle agitation on a rocking platform.
  3. Pour off the water in the sink.
  4. Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform.
  5. Pour off the SimplyBlue SafeStain in the sink.
  6. Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform.
  7. Pour off the water in the sink.
  8. brightfield grayscale image of Coomassie-stained protein gel
    Figure 1
  9. Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform.
  10. Pour off the water in the sink.
  11. Take a brightfield image of the gel with an appropriate imaging system.
    • Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to the heavy chain (HC) and light chain (LC) proteins, respectively. There should be very little background staining.

    *Pro-Tip* A large shift in the electrophoretic mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed correctly in the cell (e.g., failure to cleave the signal peptide) and may not be functional. This sample would fail Addgene QC and would not be used.

Section 3: Image Analysis

  1. Use ImageJ or a similar photo software to determine the relative intensity of the protein bands to the overall lane.
  2. Import the gel image into ImageJ.
    • Select File.
    • Select Open.
    • Choose the location of the file to open.
  3. Change the image type to 8-bit.
    • Select Image.
    • Select Type.
    • Choose 8-bit.
  4. Determine each lane of the gel as follows:
    • Three panels of Coomassie-stained protein gel in photo software with yellow boxes around lane 1 (first panel), lane 1 and 2 (second panel), and every lane (third panel).
      Figure 2
    • Using the box tool, draw a box around the entire first gel lane (as in Figure 2).
    • Select Analyze.
    • Select Gels.
    • Select Select First Lane.
    • Drag the box to the next lane.
    • Select Analyze.
    • Select Gels.
    • Select Select Next Lane.
    • Repeat for all lanes.
  5. Plot the area under the curves for the protein bands as follows:
    • Select Analyze.
    • Histogram of protein band intensity with yellow lines outlining the area under the curve.
      Figure 3
    • Select Gels.
    • Select Plot Lanes.
  6. One graph per lane will appear with peaks representing each protein band.
  7. Use the line tool to connect the bottom of each peak.
  8. Select the wand tool.
  9. Fill in each peak by selecting the center of the peak.
  10. Export results as a csv file.
    • In the results table, select File and Save As.
  11. Determine the percent purity of the samples.
    • % purity = (Area of HC + Area of LC) / (Sum of the Area of lane bands) x 100
    • Example for AR0018 (lane 2 in Figure 1):
    Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC) Peak 4 (LC) Total Area HC + LC Area % Purity
    AR0018 310.142 207.971 11469.296 4655.113 16642.522 16124.409 96.88681199

    *Pro-Tip* Addgene uses recombinant antibody preps that have a purity of ≥90% but some labs use unpurified tissue culture supernatant routinely without any issues.

  12. Plot the mg/mL versus the HC + LC Area for the IgG standards in Microsoft Excel.
  13. Add a linear trendline for the standards.
    • Microsoft Excel can calculate this for you as follows:
      • Select the graph.
      • Select the Chart Design tab.
      • Select the Add Chart Element drop down menu and select Trendline, Linear.
    • Determine the linear equation and the R2 of the trendline on Microsoft Excel as follows:
      • Select the graph.
      • Select the Chart Design tab.
      • Select the Add Chart Element drop down menu and select More Trendline Options.
      • Select Display Equation on chart.
      • Select Display R-squared on chart.
      • *Pro-Tip* The R2 of the trendline should be between 0.95 - 1.

  14. Use the equation of the trendline to determine the concentration of your samples based on your sample’s HC + LC area.
    • Example calculation:
      • If the sample HC + LC Area is 18179.442 and the linear equation of the standard curve is y = 14725x + 3748.4, then substitute 18179.442 for y and solve for x.
        • 18179.442 = 14725x + 3748.4
        • x = 0.980
      • The concentration of the sample is 0.980 mg/mL