How to Stain an SDS-PAGE gel

Summary

This video tutorial walks through three methods for staining gels used in electrophoresis: Page Blue, Kasasi, and Silver Staining. The Page Blue method is quick and efficient, requiring some microwave heating. Kasasi offers a slower, more traditional approach without the need for microwaving. Silver staining, the most sensitive method, uses hazardous chemicals and involves a multi-step process for optimal results. Each method has its benefits, with Page Blue being the fastest, Kasasi more time-consuming but simpler, and Silver providing greater sensitivity at the cost of complexity.

Takeaways

• 😀 Always unplug the electro and remove the lid before disassembling the SDS page setup.
• 😀 Carefully pour out the buffer from the chamber and remove the glass plates, as they are fragile.
• 😀 Use distilled water to rinse the glass plates, as water helps lubricate and ease the separation of the gel.
• 😀 The stacking gel can be removed if desired, but be cautious not to tear the resolving gel when doing so.
• 😀 If the gel doesn't come off easily, try lifting the corner and adding water to separate it from the plate.
• 😀 Wash the gel a few times with distilled water before microwaving the gel for staining.
• 😀 After microwaving for a minute, shake the gel on a shaker for 5 minutes to allow dye absorption.
• 😀 Remove excess water from the gel before microwaving again for 30 seconds to help the dye absorb fully.
• 😀 To speed up the staining process, you can use King WIP wipes to absorb excess dye from the solution.
• 😀 Three main staining methods are covered: Page Blue (quick), Kasasi (older and slower), and Silver Staining (sensitive but slow).

Q & A

What is the first step after finishing your SDS-PAGE run?

The first step is to unplug the electrophoresis unit, remove the lid, and carefully take out the inner chamber. You should then pour out the buffer from the chamber.

Why is it important to handle the glass plates carefully when disassembling the gel setup?

The glass plates are very fragile and can break easily, so they must be handled with care during the disassembly process.

What should you do before microwaving the gel for staining with Page Blue?

Before microwaving the gel, fill a microwavable container halfway with distilled water and rinse the glass plates. This helps lubricate the plates and makes it easier to separate the gel from the plates.

Should you remove the stacking gel when preparing for staining, and why?

It's optional to remove the stacking gel. However, it is recommended to remove it carefully to avoid damaging the resolving gel.

What is the purpose of microwaving the gel with Page Blue staining solution?

Microwaving the gel helps heat up the dye, which facilitates its absorption into the gel. The heating also helps speed up the staining process.

How do you ensure that the Page Blue dye is effectively absorbed into the gel?

After microwaving the gel for 30 seconds, you should shake the container on a shaker for about 10 minutes. If the dye is not fresh, shaking for a longer time can improve staining results.

What should you do to remove excess dye from the gel?

After staining, you should remove excess dye by washing the gel with distilled water and ensuring any excess dye is drained off. This helps prevent dilution of the dye and improves staining quality.

How does the King Wipe trick help in the staining process?

The King Wipe trick involves using two pieces of King wipes to absorb the excess dye in the staining solution, which speeds up the staining process.

What are the steps for staining a gel using Kasasi?

To stain a gel with Kasasi, first immerse the gel directly into the Kasasi stain. The staining time is typically between 20 and 60 minutes. After staining, transfer the gel into a solution of methanol and acetic acid to complete the staining process.

Why is silver staining considered more sensitive than Page Blue and Kasasi?

Silver staining is more sensitive because it involves a more complex series of steps, including gel fixation, oxidation, silver nitrate staining, and development. This method allows for the detection of even low-abundance proteins, which Page Blue and Kasasi may not reveal as effectively.