Nov 04, 2022
Coomassie Purity Stain of Recombinant Antibodies
- 1Addgene
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2022. Coomassie Purity Stain of Recombinant Antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59255g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 05, 2022
Last Modified: November 04, 2022
Protocol Integer ID: 60329
Keywords: recombinant antibodies, purification, Simply Blue
Disclaimer
We have listed 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.
Abstract
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.
Guidelines
Workflow Timeline
- Day 1: Run SDS-PAGE and stain gel
- Day 1 or later: Image analysis
Materials
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 and Consumables:
- Purified recombinant antibody, 0.9–1.1 mg/mL
- SimplyBlue SafeStain, Thermo Fisher, LC6060
- 4-12% NuPage Novex bis-tris mini gel, 1 mm thick, 10-well, Invitrogen NP0321BOX
- 20x MOPS SDS running buffer, Invitrogen NP0001
- 4x NuPage sample buffer, Invitrogen NP0007
- 10x NuPage sample reducing agent, Invitrogen NP0009
- PageRuler Plus Prestained protein ladder, Invitrogen 26619 (optional)
- Microcentrifuge tubes, Neptune 3745.X
- Pipette tips, 1000 µL, VWR 76322-154
- Pipette tips, 10 µL, VWR 76322-132
- Pipette tips, 200 µL, VWR 76322-150
- Pipette tips, 20 µL, VWR 76322-134
- Pipettes, 10 mL, VWR 89130-898
- Pipettes, 25 mL, VWR 89130-900
- Pipettes, 5 mL, VWR 89130-896
- Pipettes, 50 mL, VWR 89130-902
- Gel loading tips, Corning 4853
- PBS, 1X pH 7.4, VWR 45000-446
- 250mL sterile bottles, Corning 430281
- Deionized water
- IgG isotype standard 2.5 mg/mL - can use commercial standard or validate an in-house standard
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 into 12 µL PBS
- 1.0 mg/mL: 20 µL of 1.5 mg/mL into 10 µL PBS
- 0.75 mg/mL: 15 µL of 1.0 mg/mL into 5 µL PBS
- 0.5 mg/mL: 13.3 µL of 0.75 mg/mL into 6.7 µL PBS
- 0.25 mg/mL: 10 µL of 0.5 mg/mL into 10 µL PBS
- 0.125 mg/mL: 10 µL of 0.25 mg/mL into 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.
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.
SDS-PAGE
SDS-PAGE
1h 10m
1h 10m
Add 5 µL of 4X sample buffer to each sample.
Add 2 µL 10X reducing agent to each sample.
Spin the sample briefly in the microcentrifuge.
Heat the samples for 00:10:00 at 100 °C in a heat block.
10m
Spin the sample briefly in the microcentrifuge.
Remove the gel from the plastic wrapper and rinse with deionized water.
Gently remove the white sticker on the bottom of the gel.
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.
Carefully remove the comb from the gel.
Rinse each well with 200 µL 1X MOPS running buffer.
Fill the chamber with 1X MOPS running buffer.
Make sure that the chamber is sealed.
Load 5 µL of the prestained protein ladder to the appropriate well.
Note
When possible, skip one lane before loading samples.
Load 20 µL of each recombinant antibody sample to the appropriate well.
Note
Leaving clear lanes between samples will make quantifying the gel easier.
Cover the electrophoresis chamber and attach to a power supply.
Run the gel at 150 V for 01:00:00 .
Note
If the samples are running unevenly and the dye front looks like a “smile”, reduce the voltage.
1h
Turn off the power supply and unplug the electrophoresis chamber.
Remove the gel from the chamber.
Use the metal spatula to gently break the gel cast open.
Use a razor blade to cut the top of the wells and bottom part of the gel where dye is visible.
Staining the Gel
Staining the Gel
3h 5m
3h 5m
Place the gel in a plastic tray with 100 mL of deionized water.
Rinse gel with deionized water for 00:05:00 with gentle agitation on a rocking platform.
5m
Pour off the water in the sink.
Add 20 mL of SimplyBlue SafeStain and incubate for 01:00:00 with gentle agitation on a rocking platform.
1h
Pour off the SimplyBlue SafeStain in the sink.
Add 100 mL of deionized water and incubate for 01:00:00 with gentle agitation on a rocking platform.
1h
Pour off the water in the sink.
Add 100 mL of deionized water and incubate for 01:00:00 with gentle agitation on a rocking platform.
1h
Pour off the water in the sink.
Take a brightfield image of the gel with an appropriate imaging system.
Note
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.
Note
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.
Figure 1: Brightfield grayscale image of Coomassie-stained protein gel
Image Analysis
Image Analysis
Use ImageJ or a similar photo software to determine the relative intensity of the protein bands to the overall lane.
Import the gel image into ImageJ.
- Select File.
- Select Open.
- Choose the location of the file to open.
Change the image type to 8-bit.
- Select Image.
- Select Type.
- Choose 8-bit.
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.
Figure 2: Coomassie-stained protein gel in ImageJ photo software.
Plot the area under the curves for the protein bands as follows:
- Select Analyze.
- Select Gels.
- Select Plot Lanes.
One graph per lane will appear with peaks representing each protein band.
Figure 3: Histogram of protein band intensity with yellow lines outlining the area under the curve.
Use the line tool to connect the bottom of each peak.
Select the wand tool.
Fill in each peak by selecting the center of the peak.
Export results as a csv file.
- In the results table, select File and Save As.
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):
| A | B | C | D | E | F | G | H | |
| 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 |
Note
Addgene uses recombinant antibody preps that have a purity of ≥90% but some labs use unpurified tissue culture supernatant routinely without any issues.
Plot the mg/mL versus the HC + LC Area for the IgG standards in Microsoft Excel.
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.
Note
The R2 of the trendline should be between 0.95 - 1.
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