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)
  • Blotting membrane - nitrocellulose or PVDF (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)
  • 4 blotting sponge pads (e.g. Invitrogen EI9052) - usually included with electroblotting unit


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 - handle materials with gloved hands. Use forceps only for the blotting membrane.

  1. Remove the gel from the mold. Ue a scalpel to trim the lanes off the top and the rough edge off the bottom. Soak in a small dish of 1x Transfer buffer “back” side up. Note: the proteins will migrate "backward" onto the nitrocellulose filter.
  2. In a small flat tupperware-like or glass container of 1x Transfer Buffer, wet two blotting sponge pads. Stack one on top of the other.
  3. Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the two wet, stacked sponge pads.
  4. Carefully place the gel carefully onto the wet blotting paper. Make sure the gel is "back" side up.
  5. IMPORTANT: Use a smooth "roller" (e.g. a clean, short piece of a plastic graduated or aspiration pipette) to roll away any bubbles that may be trapped between the blotting paper and gel. Note, bubbles will block protein transfer.
  6. Take the blotting membrane and cut off a small piece of one of the corners with a pair of scissors.
  7. Wet the blotting membrane in the 1x Transfer buffer. Orient the cut corner so that it is on the bottom right. Carefully and gradually (starting at one edge) lay it on top of the gel (to avoid bubbles).
  8. Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the blotting membrane.
  9. IMPORTANT: Use a smooth "roller" (e.g. a clean, short piece of a plastic graduated or aspiration pipette) to roll away any bubbles that may be trapped between the gel and blotting membrane. Note, bubbles will block protein transfer.
  10. Wet the remaining two blotting sponge pads. Stack these on top of the blotting paper.

REMEMBER: From bottom to top, it's Sponge-Sponge-Paper-Gel-Membrane-Paper-Sponge-Sponge


Prepare the electrophoresis set-up

  1. Place the gel and filter stack into the elctroblotting module/holder so that the membrane is closest to the positive electrode side and the gel is closets to the negative electrode side.
  2. Insert this assembly into the electrophoresis tank with the positive and negative electrode sides in the proper orientation. Note: If using the Invitrogen XCell SureLock system, use the clamp to secure the blotting module.
  3. Fill the tank with the appropriate solution(s)
    1. XCell SureLock: fill the inner chamber with 1x Transfer Buffer. Fill the outer chamber with deionized water.
    2. Other systems: typically 1x Transfer Buffer for the whole tank, plus a frozen ice pack to keep the system cooled down.


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


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