Protocols
Coomassie Purity Stain of Recombinant Antibodies

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: June 14, 2023

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

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 10 min at 100 °C in a heat block.
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.
Pro-Tip
When possible, skip one lane before loading samples.
Load 20 µL of each recombinant antibody sample to the appropriate well.
Pro-Tip
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 1 h.
Pro-Tip
If the samples are running unevenly and the dye front looks like a “smile”, reduce the voltage.
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.

Section 2: Staining the Gel

Place the gel in a plastic tray with 100 mL of deionized water.
Rinse gel with deionized water for 5 min with gentle agitation on a rocking platform.
Pour off the water in the sink.
Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform.
Pour off the SimplyBlue SafeStain in the sink.
Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform.
Pour off the water in the sink.
Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform.
Pour off the water in the sink.
Take a brightfield image of the gel with an appropriate imaging system.

Figure 1
- 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

Use ImageJ (Link opens in a new window) 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:

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.
Plot the area under the curves for the protein bands as follows:

Figure 3
- Select Analyze.
- Select Gels.
- Select Plot Lanes.
One graph per lane will appear with peaks representing each protein band.
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: $$P={(H + L)\over A}*100$$

Where:

P = Purity, %
H = Area of HC peak
L = Area of LC peak
A = Total area of all bands in the lane

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	96.887%

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