2022-10-28
SDS-PAGE and Western Blot
Protein Sample Preparation
Protein samples from different stages of purification (Periplasmic Extract, Flow Through, Wash, and Elution) were prepared.
The samples were mixed with reducing sample buffer and boiled to denature the proteins.
The gel was set up in the electrophoresis chamber and immersed in buffer.
Samples were loaded, and the gel was run to separate proteins based on size.
Once the run was complete, the setup was disassembled, and the gel was transferred for staining or blotting.
Coomassie Staining
The gel was stained with Coomassie blue to visualize the separated proteins.
The gel was destained to remove excess dye, leaving visible protein bands.
Western Blot - Protein Transfer
Proteins from the gel were transferred to a nitrocellulose membrane using an electric current in a transfer tank.
Protein Detection (Western Blot)
The membrane was blocked to prevent non-specific binding.
The membrane was incubated with a primary antibody targeting the protein of interest.
The membrane was washed and incubated with a secondary antibody that will help visualize the protein.
Visualization
The membrane was developed using a detection solution that produces color in the presence of the enzyme linked to the secondary antibody.
The blot was compared with the Coomassie-stained gel to analyze the presence and purity of the target protein.
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