Western Blot - wet transfer

Updated 6/25/2016 08:13 PM

by Haynes Lab (created by Karmella)

Introduction

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 [250mM Tris; 1.92M glycine; pH approx. 8.3] (see recipe)

Methanol

Blotting membrane - nitrocellulose or PVDF (e.g., Bio-Rad Nitrocellulose & filter sets 1620212)

Cut to the size of the gel

Blotting paper

Four pieces cut to the size of the membrane & gel

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 [12.5mM Tris; 96mM glycine; 10% methanol]

Add ~200mL dH₂O, 25mL Tris-glycine concentrate, and 50mL methanol to a 500mL bottle. Swirl to mix.

Fill up to 500mL with dH₂O (+ ~225mL 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.

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.

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.

Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the two wet, stacked sponge pads.

Carefully place the gel carefully onto the wet blotting paper. Make sure the gel is "back" side up.

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.

Take the blotting membrane and cut off a small piece of one of the corners with a pair of scissors.

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).

Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the blotting membrane

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.

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

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.

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.

Fill the tank with the appropriate solution(s)

XCell SureLock: fill the inner chamber with 1x Transfer Buffer. Fill the outer chamber with deionized water, about halfway.

Other systems: typically 1x Transfer Buffer for the whole tank, plus a frozen ice pack to keep the system cooled down.

Connect the electrodes to the chamber and a power supply.

Run the transfer...

Invitrogen XCell SureLock - 30 volts for 1 hour.

Obtain a small dish of deionized water (a bit larger than the membrane).

Once the transfer is complete, unplug the leads and disassemble the gel/membrane/filter stack. Remove the membrane and place it into the dI-water. You should see that the loading dye (and pre-stained ladder) is now absent fromt he gel and present on the membrane.

Continue with the protocol "Western blot - antibody staining."