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
• 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 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 (+ ~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.
3. Add protein samples to 1.5 mL tubes. Dilute the stock proteins in an appropriate buffer. Make sure the protein sample volume = the final volume - (concentrated sample buffer volume + 1.0 μL 1M DTT)
   1. Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
   2. Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
4. Calculate the number of lanes used = protein samples + protein standard(s). If this is less than the total number of lanes, prepare "dummy" samples (sample buffer + DTT + water) to fill empty wells
5. Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp.

Prepare the electrophoresis chamber

1. Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer.
   1. Note: 500 mL is sufficient for a mini gel system. For a larger chamber, make 1 L of 1x buffer.
2. Remove strip from the bottom of the gel to expose it.
3. Secure the gel, exposed side facing the outer wall, inside the chamber. Note: If running a single gel, set up a dummy gel mold opposite the actual gel.
4. Use the 1x running buffer to fill the inner camber to the top (above the gel lanes). For a mini gel system, this will take about 200 mL.
5. IMPORTANT: If using NuPAGE buffer, add 500 μL Antioxidant into the buffer in the inner chamber drop-wise, evenly over the surface of the buffer.
6. Next, fill the outer chamber just so that the bottom of the gel is submerged.
7. Gently pull out the well comb and discard it.
8. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
9. Run the gel @ 120 V until the dye front reaches the very bottom.

HELPFUL RESOURCES

• Gel setup: Video - Running the Gel on the XCell SureLock® system