Protein Staining in Polyacrylamide Gels with Rapid and Sensitive Colloidal Coomassie G-250

Summary

This video script outlines a modified Kumasi staining protocol for detecting proteins in polyacrylamide gels, using two-dimensional gel electrophoresis as an example. It details the process of isoelectric focusing and SDS-PAGE, emphasizing the protocol's high sensitivity, cost-effectiveness, and practicality for analytical proteomics. The script guides through the preparation of reagents, staining, and destaining, highlighting the importance of using high-quality chemicals and thorough washing for optimal results.

Takeaways

• 🧪 Modified Kumasi staining protocol is demonstrated for detecting proteins in polyacrylamide gels.
• 🎯 The example of two-dimensional gel electrophoresis is used to illustrate the procedure.
• 🌟 The first dimension involves isoelectric focusing (IEF) using immobilized pH gradient (IPG) strips.
• 🥼 Protein samples are applied via anodic cup loading during the focusing process.
• 🧫 The second dimension is performed by SDS-PAGE for separation based on molecular weight.
• 💧 After electrophoresis, proteins are detected by staining with colloidal Kumasi.
• 👩‍🔬 Nadine Dubala from the University of Dusseldorf emphasizes the protocol's advantages in sensitivity, cost, and practicality.
• 🔬 The method is particularly useful in analytical proteomics for myocardial protein expression analysis.
• 🛠️ Proper preparation of the staining solution is crucial, with sequential addition of components.
• 🧼 Efficient washing of gels post-SDS-PAGE is necessary to remove residual SDS for effective staining.
• 📈 The protocol can detect as low as one nanogram of protein, making it a valuable tool for proteomics research.

Q & A

What is the primary focus of the video?

The primary focus of the video is to demonstrate the power of a modified Kumasi staining protocol for detecting proteins in polyacrylamide gels, specifically using the example of two-dimensional gel electrophoresis.

What is the first dimension separation in 2D gel electrophoresis?

The first dimension separation in 2D gel electrophoresis is isoelectric focusing (IEF), where immobilized pH gradient (IPG) strips are used to separate proteins according to their isoelectric points.

How are the IPG strips prepared for protein sample application?

The IPG strips are prepared by rehydrating them individually in 125 microliters of rehydration solution using the Immobiline dry strip re-swilling tray for at least 10 hours.

How are the protein samples prepared for the IEF?

Protein samples are prepared by dissolving TCA-precipitated protein samples from different heart preparations in IEF sample buffer to a concentration of 100 micrograms protein per 100 microliters, with solubilization taking at least 30 minutes at room temperature on a vortexer.

What is the role of the cooling unit during IEF?

The cooling unit is set to 20 degrees Celsius to help maintain a consistent temperature throughout the IEF process, which is crucial for the proper separation of proteins.

How are the proteins separated in the second dimension of 2D gel electrophoresis?

The second dimension separation is performed by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) on a vertical electrophoresis system, where proteins are separated for 2.5 hours starting at 80 Volts for 15 minutes, followed by 120 Volts until the dye front reaches the bottom of the gel cassette.

What are the critical steps in preparing the colloidal Kumasi staining solution?

The critical steps include dissolving aluminum sulfate, intermixing ethanol and adding CBB G250, followed by the addition of orthophosphoric acid to aggregate the Kumasi molecules into their colloidal state, and finally adjusting the volume with Millie Q water without filtering.

Why is it important to wash the gel thoroughly before staining?

Thorough washing of the gel is important to remove residual SDS, which can disrupt the binding of the dye to the protein and cause poor sensitivity in the staining process.

What is the recommended incubation time for the Kumasi staining to achieve close to 100% staining?

While 80 to 100% of the staining can be accomplished within two hours of incubation, an overnight incubation is recommended to achieve close to 100% staining.

How does the sensitivity of the Kumasi staining compare to other staining methods?

The Kumasi staining is comparable to silver staining, which is reported to be of high sensitivity and compatibility with mass spectrometry, making it a good alternative for non-labeling methods in gel-based proteomics.

What are the key factors to consider when using the Kumasi staining procedure?

Key factors include using analytical grade chemicals and water for purity, the sequential addition of components to the staining solution, and efficient washing of residual SDS from the gels after electrophoretic separation to ensure optimal results.