Hi, ZaiYong

SDS-PAGE can also separate high MW protein of less than 500KD. But when you prepare the gel, the Tris-Glycine gel should be replaced by the 3%-8%Tris-Acetate gel. and agarose gel isn't common usage for separate protein.

Thank you for your helpful suggestion! Based on your advice, I have successfully located a relevant protocol in a research article. We plan to implement this method in our experiments. Here is the reference to the article: https://pubmed.ncbi.nlm.nih.gov/22585488/

Hi, Dr. Zheng, I had also looked into the experimental methods of high molecular weight gel electrophoresis before. Here are the reference literature and experimental procedures that were used at the time for your reference.

Article Disease proteomics of high-molecular-mass proteins by two-di...

High Molecular Weight Protein Electrophoresis Method (Agarose 2-DE)

1. Sample Preparation

Protein Extraction

• Use 7 M urea + 2 M thiourea as a solubilizing agent to facilitate the dissolution of high-molecular-weight (HMM) proteins.
• Alternatively, a 2% SDS extraction buffer (containing 10% glycerol, 5% β-mercaptoethanol, 0.0625 M Tris-HCl, pH 6.8) can be used to enhance HMM protein solubility.

Ultracentrifugation

• Perform ultracentrifugation at 112,000 × g (Beckman Coulter TLA-55 rotor, 50,000 rpm, 20 min) to remove DNA and other insoluble materials, reducing streaking in gel electrophoresis.

2. Agarose Gel Isoelectric Focusing (IEF, First-Dimension Electrophoresis)

• Use 1% agarose as the gel matrix and conduct isoelectric focusing (IEF) for protein separation.
• Agarose IEF allows better migration of HMM proteins compared to polyacrylamide IEF due to its larger pore size, making it more suitable for HMM proteins.
• After IEF, the gel must be equilibrated to remove SDS interference and ensure compatibility with subsequent SDS-PAGE.

3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE, Second-Dimension Electrophoresis)

• Use 1% SDS in both the stacking and separation gels instead of the conventional 0.1% SDS to improve HMM protein solubility during PAGE, preventing protein precipitation or retention in the first-dimension gel.
• This step enhances the clarity of electrophoretic bands and improves the detection sensitivity of HMM proteins.

4. Protein Staining and Identification

• Use Coomassie Blue R staining for protein visualization.
• Silver staining or 2D-DIGE (fluorescence-based two-dimensional difference gel electrophoresis) can be used for increased protein detection sensitivity.
• Combine with mass spectrometry (MS) for protein identification, particularly for cancer proteomics and disease research.

Hope this helps u! Welcome to discuss any follow-up details. If you have any questions about the reagents used in the electrophoresis process, feel free to consult me.