Protein Apparatus
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Transcript of Protein Apparatus
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Protein Apparatus
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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
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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
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We will use premade gels: they are difficult to pour and contain toxic components
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Remember to wear gloves when handling gels
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Remove gel from box and tear side of wrapping
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Remove gel from protective pouch
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You need to pull and remove the strip at the bottom of the gel
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Remove the comb from the gel(after the apparatus is set up)
and wash out the wells
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Remove the comb from the gel
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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
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Final setup of gels
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Make sure the wells are facing inward
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2 gels should be put into the apparatus
• Buffer can then be poured in between the 2 plates
• Notice the black and red electrodes
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Place the entire gel apparatus into the tank
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You may want to use longer/thinner tips to load the gel
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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
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Another picture of gel loading
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The final step
• Put the cover on in the correct orientation
• Red to red and black to black
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Gel after staining with Coomassie Blue
• Standards Measured in kD
• Fig. 7.12
• 10 kD – 250 kD