Protein Gel

Getting Started

You have samples and you want to see what proteins are in that sample and how much of each protein there is. You can use this protocol to semi-quantitate protein sizes and concentrations in your samples.

Materials and Equipment

Protocol

Denature samples.

Label one tube per sample.

Add 7 uL of 4x sample buffer to each tube.

Add 21 uL of each sample to its tube. Mix by pipetting.

Incubate samples at 90C / 10 min.

Set up gel box.

Open an individually wrapped protein gel (we use 10% - 20% Tris Gly gels). Throw out the bag that the gel came with.

Remove plastic tape at bottom of gel. This exposes a strip of gel to buffer, allowing current to flow through the gel and out the bottom.

Place gel in gel box and seal tightly.

Pour running buffer (we use Tris MOPS) into the front half of the gel box reservoir. Check to make sure that the gel is tightly sealed (i.e., no buffer can run from the front to the back of the gel box).

Pour running buffer into the back half of the gel box reservoir.

Remove comb from top of gel.

Load between 10 uL and 20 uL of sample onto each lane of the gel. Remember to include a protein ladder!

Run the gel. We use 150 V / 60 min.

Stain and destain the gel.

Using a gel knife (looks like a paint can opener), pop open the plastic cassette holding the gel.

Poke the gel out of the plastic box by pushing the gel knife through the exposed strip of gel on the back of the plastic cassette. This step is easier if the gel knife is wet.

Briefly rinse the gel in water (milliQ).

Submerge gel in a rapid staining protein stain in a waterproof container. Incubate gel at room temperature with rocking for up to 1 hr.

Decant staining solution, rinse gel at least once, then submerge gel in water (milliQ). Incubate gel at room temperature with rocking for 15 min.

Repeat above step until bands are clearly visible (we do three destaining steps)

Image gel.

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Note: use an imager to semi-quantitate!