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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. - PowerPoint PPT Presentation
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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