Handout 3
Experiment: SDS gel electrophoresis analysis of pig heart lactate dehydrogenase (LDH)
Capsule summary: Load SDS PAGE. Run gels and stain with Coomassie Blue. Remove stain and add destain
Materials:
BioRad gel electrophoresis equipment
Prepared samples to load from previous lab
Power supply
Plastic containers for staining
Coomassie Blue stain
Destain
Platform shaker
Reagents
Staining Solution
0.15 g Coomassie Brilliant Blue
200 mL methanol
Stir until dissolves, then add 35 mL acetic acid
Fill upto 500 mL with d H2O
Destaining Solution
400 mL methanol
70 mL acetic acid
Fill up to 1L with d-H2O
5X Running buffer (prepare 1 L for class)
15 g Tris Base
72 g glycine
5 g SDS
Fill to 1 L with d-H2O
Store at 4°C
Procedure:
Overall procedure: Load gels with P20 pipetman. Run at 120 V until dye front reaches bottom of gel. Remove gel
to staining solution and subsequently to destaining solution. Wash equipment and put away.
Detailed procedure:
Place samples in rack in order of loading (left to right). Assemble gels into cassettes (two gels per cassette). Place
the cassettes into the electrophoresis tank. Add 1X Running buffer to top (inside) reservoir to above the lower
glass plate. Make sure there are no leaks. Add Running buffer to outside reservoir to appropriate level depending
on the number of cassettes loaded. Load samples left to right. First sample (1 st lane) is always 5-7 µL of protein
loading standards. Continue to load samples into lanes 2 through 10. Put lid on tank, attached cables with
appropriate polarity into power supply. Power up the power supply to 120 V. Check for bubbles emanating from
platinum wires in the cassettes (both inside reservoir and outside reservoir). If no bubbles recheck connections.
Once tracking dye reaches bottom of gel turn off power supply. Carefully separate glass plates and place gel in ~50
mL of Coomassie stain. Stain with shaking for 15 min. Remove stain and replace with ~50 mL of destain. Leave on
shaker overnight.
Watch youtube video for loading and running gels: https://www.youtube.com/watch?v=XUjLO-ek2C8&t=5s
Note: at the start of next lab period, remove destain and replace with ~100 mL water. After 15 min students can
take picture of gel. A lightbox is helpful for detecting the bands.
Possible active learning session.
You can divide the white board with lines so that groups can simultaneously draw on the board. Each group should
be able to draw what the gel should look like once it is stained. This activity is useful during the time the gels are
running.