Apr 15, 2026
Protein quantification by SDS-PAGE and Coomassie staining
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Protein quantification by SDS-PAGE and Coomassie staining. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qqqev25/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: December 13, 2024
Last Modified: April 15, 2026
Protocol Integer ID: 115392
Keywords: ASAPCRN, Immunoblotting, Western blot, ATP13A4, protein quantification by sd, protein quantification, accurate protein quantification, protein concentration by sd, quantifying protein concentration, data analysis for accurate protein quantification, protein separation via electrophoresi, protein separation, electrophoresi, protein
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol provides a step-by-step guide for quantifying protein concentration by SDS-PAGE and Coomassie staining. It details the preparation of samples and BSA standards, protein separation via electrophoresis, staining, imaging, and data analysis for accurate protein quantification.
Materials
Materials
- β-mercaptoethanol (Cat# M3148, Sigma)
- BSA (Cat# A4161, Sigma)
- NuPAGETM LDS Sample Buffer (4X) (Cat# NP0007, Invitrogen)
- NuPAGETM 4–12% Bis-Tris precast gels (Cat# NP0321, Cat# NP0322, Cat# NP0322; Invitrogen)
- NuPAGETM MOPS SDS Running Buffer (20X) (Cat# NP0001; Invitrogen)
- SeeBlueTM Plus2 Pre-stained Protein Standard (Cat# LC5925, Invitrogen)
- InstantBlue‱ Coomassie Protein Stain (ISB1L) (Cat# ab119211, abcam)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Protein separation by gel electrophoresis
1h 5m
Prepare protein samples (12 µL ) with 4 µL LDS sample loading buffer (supplemented with β-mercaptoethanol).
Prepare BSA standards (12 µL of 0.01 mg/ml, 0.02 mg/ml, 0.04 mg/ml, 0.08 mg/ml, 0.1 mg/ml) with 4 µL LDS sample loading buffer (supplemented with β-mercaptoethanol).
Load prepared standards and protein samples onto a precast NuPAGE 4-12% BisTris gel, alongside molecular weight markers for reference.
Run the gel for 01:00:00 at 170 V, Room temperature . Use MOPS running buffer.
1h
Coomassie staining
1h
Stain the gel with Coomassie for approximately 01:00:00 or until bands are clearly visible.
1h
Destain the gel using distilled water until the background is clear and bands are distinct.
Imaging and analysis
Capture an image of the stained gel using an ordinary scanner or Bio-Rad ChemiDoc MP imaging system.
Plot the intensity of the BSA bands as a function of protein concentration. Generate a standard curve using these data points.
Compare the intensity of the protein bands to the standard curve. Calculate the concentration of the protein of interest based on its band intensity and the curve.
Note
Ensure that the protein sample is within the linear range of the standard curve for accurate quantification.