Team:StanfordBrownSpelman/Lab Techniques6

Working with Protein Gels

What to Know and Do

PAGE Gel Preparation, Running, and Scanning
(proteins only)

Setting up and Running the Gel
1. Use NuPAGE (NOT Bolt) gels, located in middle room fridge on top shelf, far left
2. Keep the gel in its case and rinse off with DW water
3. Carefully remove comb from the case by pulling out from both sides, be gentle! Also remove white tape on bottom for current circulation when gel is running
4. Keep gel in its case. Load gel into running box (upright). Make sure gel is secure and the segment for loading the wells is on the side opposite you.
5. Add SDS running buffer (not MOPS!) so wells overflow into front of the box
6. Before loading must wash out each gel well by pipetting gently up and down
7. Prepare loading samples (also refer to gel kit instructions)
      a) 7.5ul of product + 2.5ul SeeBlue loading dye in each well
      b) 6ul SeeBlue NuPAGE ladder (purple top, keep refrigerated)
8. BEFORE LOADING SAMPLES HEAT THEM for 10 minutes at 70 C
9. Load gel with ladder and dyed samples

NOTE: When you load a PAGE gel push pipette against the front of the box. The gel has 12 wells, if you do not need to use all 12, then avoid using the very first and the very last well; as the gel runs the current pulls unevenly from the sides (creates “smiling effect” that can make interpreting the scan more difficult)

10. Run gel for 35 minutes at 150-200V

Fixing and Staining the Gel

1. After running, the gel needs to be fixed, stained overnight, and then washed before it can be scanned on the Typhoon scanner. Prepare fixing solution for the gel (ideally you should do this while the gel is running)

Fix solution recipe: 50% methanol, 7% acetic acid, fill with milliQ water to 200ul
   
2. Remove the gel from the running case and place it in a clean container with fixing solution    
3. Put gel in 100ul of fix solution and shake in RT at 80rpm for 30 minutes    
4. Repeat step 3 with remaining 100ul fix solution    
5. Remove all fix solution from container with the gel    
6. Soak gel in 60ml SYPRO Ruby gel stain, shake overnight at 80rpm in RT.

Washing and Scanning the Gel

1. Remove PAGE gel from overnight staining and put into new, clean container
2. Wash gel in 100ul of wash solution for 30 minutes in 70-80rpm for 30 minutes

Wash solution recipe:
10% methanol, 7% acetic acid, fill with milliQ water until 100ul

3. Remove gel from wash and rinse twice with DI water for 5 minutes to remove all wash to prevent damage to scanner

Scanning a Protein PAGE gel

1. Same as DNA gel, specify size of the gel for the scanner. When loading, make sure to be gentle with the gel (it’s fragile!) and carefully separate combs when on the scanner so you can tell which well is which

      

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