Haynes:WestBlot

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Western Blot

by Karmella Haynes, 2015

Principle: Proteins that have been separated by size on a polyacrylamide gel (by PAGE)are transferred onto a solid paper-like membrane, such as nitrocellulose. One specific protein is visualized by incuabating the membrane with labeled antibodies, then imaging the membrane. A visible band will appear that corresponds to the size of the protein.

MATERIALS

• PAGE gel containing electrophoresed protein samples
• Tris-glycine concentrate [250 mM Tris; 1.92 M glycine; pH approx. 8.3] (see recipe)
• Methanol
• Blotting paper/ "filter" paper (e.g. Bio-Rad Nitrocellulose & filter sets 1620212)
• Nitrocellulose or PVDF membrane (e.g., Bio-Rad Nitrocellulose & filter sets 1620212)
• 10x PBS
• Tween-20 (e.g., G-Biosciences 786-517)

EQUIPMENT

• Electroblotting chamber (e.g. Invitorgen XCell SureLock)

PROCEDURE

Prepare 1x Transfer Buffer [12.5 mM Tris; 96 mM glycine; 10% methanol]

1. Add ~200 mL dH₂O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
2. Fill up to 500 mL with dH₂O (+ ~225 mL dH₂O). Note: deionized water from the tap is sufficient.

Prepare a gel and filter stack
Instructions for a wet chamber

1. Remove the gel from the mold, trim the top and bottom, and soak in Western blot transfer buffer “back” side up.
2. In a small flat container of 1x Transfer Buffer, wet two sponges, two pieces of blotting paper, and a nitrocellulose membrane.

Prepare the electrophoresis chamber

Prepare 1x PBS-tween [? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]

1. Add ~200 mL dH₂O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
2. Fill up to 500 mL with dH₂O (+ ~249.5 mL dH₂O). Note: deionized water from the tap is sufficient.

HELPFUL RESOURCES

• Electroblotting wet transfer: Video - How to perform a traditional wet protein transfer using the XCell SureLock® Blot Module