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Protein Apparatus

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.

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Protein Apparatus

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  1. Protein Apparatus

  2. 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

  3. 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

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

  5. Remember to wear gloves when handling gels

  6. Remove gel from box and tear side of wrapping

  7. Remove gel from protective pouch

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

  9. Remove the comb from the gel(after the apparatus is set up)and wash out the wells

  10. Remove the comb from the gel

  11. 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 Setting up the gel

  12. Final setup of gels

  13. Make sure the wells are facing inward

  14. Buffer can then be poured in between the 2 plates Notice the black and red electrodes 2 gels should be put into the apparatus

  15. Place the entire gel apparatus into the tank

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

  17. Make sure you find the wells Careful place the tip as far down as possible Load the well Remember, the wells are in the back of the gel

  18. Another picture of gel loading

  19. Put the cover on in the correct orientation Red to red and black to black The final step

  20. Gel after staining with Coomassie Blue • Standards Measured in kD • Fig. 7.12 • 10 kD – 250 kD

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