Edvotek Kit #303 - Student Module III - Purification of GFP by Column Chromatography

Summary

This instructional video guides viewers through the process of purifying Green Fluorescent Protein (GFP) using column chromatography. It outlines the necessary materials, including microtiter plates and elution buffers, and details each step—from packing and equilibrating the chromatography column to collecting and analyzing fluorescent fractions. The procedure emphasizes careful monitoring of the GFP elution process and the identification of the brightest fractions for further analysis. This engaging exploration of biotechnology equips viewers with practical skills for molecular purification, setting the stage for deeper analysis in subsequent modules.

Takeaways

• 😀 The module focuses on the purification of Green Fluorescent Protein (GFP) using column chromatography.
• 🧪 Users need various equipment, including a microtiter plate, elution buffer, and chromatography column for the procedure.
• 📏 The column must be mounted vertically on a ring stand to ensure accurate packing and elution.
• 🔄 The molecular sieve matrix must be thoroughly mixed before being added to the column to ensure proper function.
• 💧 A thin layer of elution buffer should always be maintained on top of the packed matrix to prevent drying.
• ⚖️ The GFP extract is slowly loaded into the column to ensure maximum absorption and prevent dilution.
• 🔍 Monitoring the elution progress with a long-wave UV light source is crucial to track the GFP separation.
• 🧪 Fractions are collected in a microtiter plate, starting from the first well and moving left to right.
• 🌈 Identification of the brightest fluorescent fractions is essential for selecting the most concentrated GFP samples.
• 📦 Proper labeling of the microcentrifuge tubes ('GFP Native' and 'GFP Denature') is vital for clarity in subsequent analysis.

Q & A

What is the main focus of Module III in the Edvotek instructional videos?

The main focus of Module III is the purification of Green Fluorescent Protein (GFP) by column chromatography using a cellular lysate from Module II.

What equipment is necessary for the column chromatography process?

Necessary equipment includes a microtiter plate, screw-top microcentrifuge tubes, elution buffer, molecular sieve matrix, a ring stand, and a chromatography column.

How is the chromatography column prepared for use?

The column is prepared by mounting it vertically, adding 1 ml of elution buffer, mixing the molecular sieve matrix, and pipetting the slurry into the column, ensuring it fills approximately 2/3 of the narrow part.

What steps are involved in loading the GFP extract onto the column?

The GFP extract is slowly loaded onto the column, allowing it to enter completely before starting the elution process with 1x elution buffer.

How is the progress of GFP monitored during elution?

The progress of GFP in the gel matrix is monitored by illuminating the column with a long-wave UV light source.

What is the purpose of collecting fractions in the microtiter plate?

Fractions are collected in the microtiter plate to isolate and analyze the GFP-containing samples based on their fluorescence levels.

How are the fractions evaluated for the presence of GFP?

The fractions are evaluated using long-wave UV light to identify which well contains the brightest levels of fluorescent proteins.

What should be done with the brightest GFP fractions collected?

30 microliters of the brightest elution should be transferred to a screw-top microcentrifuge tube labeled 'GFP Native' and an additional 30 microliters to another tube labeled 'GFP Denature.'

What is the next step after the purification of GFP?

After purification, the next step is Module IV, which involves the analysis of GFP by SDS Gel Electrophoresis.

Why is it important to prevent the chromatography column from running dry?

Preventing the column from running dry is crucial because it can lead to poor separation and reduced yield of the target protein.