TITLE: Coomassie Purity Stain of Recombinant Antibodies
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
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.

[BEFORE_START]
See the Materials section for preparation of necessary reagents.

Warm the hot plate to 100 °C
Thaw IgG standard and prestained protein ladder on ice.


[GUIDELINES]
Workflow Timeline
Day 1: Run SDS-PAGE and stain gel
Day 1 or later: Image analysis

[STEPS]
SECTION: SDS-PAGE
1. Add 5 µL of 4X sample buffer to each sample.

SECTION: SDS-PAGE
2. Add 2 µL10X reducing agent to each sample.

SECTION: SDS-PAGE
3. Spin the sample briefly in the microcentrifuge.
SECTION: SDS-PAGE
4. Heat the samples for 10 min at 100 °C in a heat block.

SECTION: SDS-PAGE
5. Spin the sample briefly in the microcentrifuge.
SECTION: SDS-PAGE
6. Remove the gel from the plastic wrapper and rinse with deionized water.
SECTION: SDS-PAGE
7. Gently remove the white sticker on the bottom of the gel.
SECTION: SDS-PAGE
8. Place the gel in the electrophoresis chamber and secure it.
 

SECTION: SDS-PAGE
9. Carefully remove the comb from the gel.
SECTION: SDS-PAGE
10. Rinse each well with 200 µL 1X MOPS running buffer.

SECTION: SDS-PAGE
11. Fill the chamber with 1X MOPS running buffer.
SECTION: SDS-PAGE
12. Make sure that the chamber is sealed.
SECTION: SDS-PAGE
13. Load 5 µL of the prestained protein ladder to the appropriate well.

 


SECTION: SDS-PAGE
14. 
Load 20 µL of each recombinant antibody sample to the appropriate well.

 


SECTION: SDS-PAGE
15. Cover the electrophoresis chamber and attach to a power supply.
SECTION: SDS-PAGE
16. Run the gel at 150 V for 60 min.

 

SECTION: SDS-PAGE
17. Turn off the power supply and unplug the electrophoresis chamber.
SECTION: SDS-PAGE
18. Remove the gel from the chamber.
SECTION: SDS-PAGE
19. Use the metal spatula to gently break the gel cast open.
SECTION: SDS-PAGE
20. Use a razor blade to cut the top of the wells and bottom part of the gel where dye is visible.
SECTION: Staining the Gel
21. Place the gel in a plastic tray with 100 mL of deionized water.

SECTION: Staining the Gel
22. Rinse gel with deionized water for 5 min with gentle agitation on a rocking platform.

SECTION: Staining the Gel
23. Pour off the water in the sink.
SECTION: Staining the Gel
24. Add 20 mL of SimplyBlue SafeStain and incubate for 60 min with gentle agitation on a rocking platform.

SECTION: Staining the Gel
25. Pour off the SimplyBlue SafeStain in the sink.
SECTION: Staining the Gel
26. Add 100 mL of deionized water and incubate for 60 min with gentle agitation on a rocking platform.

SECTION: Staining the Gel
27. Pour off the water in the sink.
SECTION: Staining the Gel
28. Add 100 mL of deionized water and incubate for 60 min with gentle agitation on a rocking platform.

SECTION: Staining the Gel
29. Pour off the water in the sink.
SECTION: Staining the Gel
30. Take a brightfield image of the gel with an appropriate imaging system.
 
 

 

  

SECTION: Image Analysis
31. Use ImageJ or a similar photo software to determine the relative intensity of the protein bands to the overall lane.
SECTION: Image Analysis
32. Import the gel image into ImageJ.

Select File.
Select Open.
Choose the location of the file to open.
SECTION: Image Analysis
33. Change the image type to 8-bit.

Select Image.
Select Type.
Choose 8-bit.
SECTION: Image Analysis
34. Determine each lane of the gel as follows:

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.

  

SECTION: Image Analysis
35. Plot the area under the curves for the protein bands as follows:

Select Analyze.
Select Gels.
Select Plot Lanes.
SECTION: Image Analysis
36. One graph per lane will appear with peaks representing each protein band.

  

SECTION: Image Analysis
37. Use the line tool to connect the bottom of each peak.
SECTION: Image Analysis
38. Select the wand tool.
SECTION: Image Analysis
39. Fill in each peak by selecting the center of the peak.
SECTION: Image Analysis
40. Export results as a csv file.

In the results table, select File and Save As.
SECTION: Image Analysis
41. 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):


 

 

SECTION: Image Analysis
42. Plot the mg/mL versus the HC + LC Area for the IgG standards in Microsoft Excel.
SECTION: Image Analysis
43. Add a linear trendline for the standards. 


SECTION: Image Analysis
43.1. 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.
        
SECTION: Image Analysis
43.2. 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.
 

SECTION: Image Analysis
44. 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