Protein Apparatus

Protein electrophoresis

• Treat with SDS before electrophoresis– Makes proteins negatively charged– Performs cell lysis– Partially denatures proteins

• Then, heat at 95 degrees to fully denature proteins

– Also, treat with b-mercaptoethanol to break disulfide bonds

Protein electrophoresis• Run vertically

• Use polyacrylamide gels

• Tris/Glycine/SDS running buffer

• Stain with Coomassie blue after electrophoresis

• Do not stain if performing Western Blot

We will use premade gels: they are difficult to pour and contain toxic components

Remember to wear gloves when handling gels

Remove gel from box and tear side of wrapping

Remove gel from protective pouch

You need to pull and remove the strip at the bottom of the gel

Remove the comb from the gel(after the apparatus is set up)

and wash out the wells

Remove the comb from the gel

Setting up the gel• Place your gel in the top

apparatus to the left– The wells face in

• After the gel is placed in, put the top apparatus into the bottom apparatus – close the locks

• You must have 2 gels: use a blank gel if you have only one gel to run

Final setup of gels

Make sure the wells are facing inward

2 gels should be put into the apparatus

• Buffer can then be poured in between the 2 plates

• Notice the black and red electrodes

Place the entire gel apparatus into the tank

You may want to use longer/thinner tips to load the gel

Remember, the wells are in the back of the gel

• Make sure you find the wells

• Careful place the tip as far down as possible

• Load the well

Another picture of gel loading

The final step

• Put the cover on in the correct orientation

• Red to red and black to black

Gel after staining with Coomassie Blue

• Standards Measured in kD

• Fig. 7.12

• 10 kD – 250 kD