Protein Purification and SDS PAGE

How does purification work on a Ni-column?

Exploits high affinity between His-tag and nickel ions, allowing for selective binding of proteins.

After binding, unbound proteins are washed away, and elution is performed with imidazole to release the His-tagged proteins.

How are proteins prepared for a SDS-PAGE?

• Denatured by SDS and reducing agent

• Add heat to denature

How does a SDS-PAGE work?

SDS-PAGE separates proteins based on their molecular weight by applying an electric field to a gel matrix. Proteins are denatured and coated with SDS, giving them a uniform negative charge, allowing them to migrate towards the positive electrode.

What does the SDS and reducing agent do?

the reducing agent breaks disulfate bonds, linearizing the protein

SDS coats the protein with uniform (-) charge, allowing for separation by size