How to Isolate and Purify Protein Bands After SDS-PAGE
After SDS-PAGE, if you want to recover a specific protein band from the gel, you can purify it using the following steps:
1. Staining and Destaining the Gel
First, stain the entire gel with an appropriate dye (such as Coomassie Brilliant Blue). After a set time, destain the gel until the desired protein band is clearly visible.
2. Cutting the Gel
Using a clean, sharp blade or scissors, cut the gel along the stained protein band, minimizing the non-protein areas around the band.
3. Protein Extraction
Place the cut gel pieces into a microcentrifuge tube. Add an appropriate extraction buffer (e.g., 50 mM Tris-HCl, pH 8.0, containing 0.1% SDS or another suitable buffer). Gently shake or vortex the gel pieces for a period to allow the protein to diffuse into the liquid. Centrifuge to collect the protein solution from the liquid, and discard the gel pieces.
4. Protein Concentration and Further Purification
Use a protein concentrator (such as Amicon or other brands of ultrafiltration devices) to concentrate the protein solution. If needed, other purification methods such as affinity chromatography, ion exchange chromatography, or gel filtration chromatography can be used to further purify or enrich specific proteins.
5. Protein Identification
To confirm the identity of the purified protein, use mass spectrometry or other biochemical analysis methods for identification.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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