SDS-page gels

SDS-page gels

Creating a Protein Gel

Materials

• Clean casting stand w/ dual gel apparatus
• Glass plate holders (w/ white or black knobs)
• Additional spacers (1.5) with (1.5) size combs
  • Different spacers can be used to create different gel widths (ie 0.75 spacers compared to 1.5 spacers)
• Two clean glass plates (one larger and one smaller, cleaned with ethanol + water)
  • If a plate has a crack on it, crack can be sealed with clear tape
  • Look out for cracks on the bottom of the plates, this will cause leaks

Steps for assembling gel apparatus

1. Place the spacers onto the larger clean glass, by the edges, and place the smaller clean glass plate on top
2. Place the two glass plates into the glass plate holder (knobs facing away from you) and adjust until bottom is flush and spacers are in the corners. Tighten the knobs
3. Take the whole glass holder (w/ adjusted plates) onto the casting apparatus. Start by pushing the holder into the rubber gasket on the bottom of the casting apparatus and snap into the top portion
4. To ensure the seal has been made, use a dropper to add a small portion of DI water to test. Test also the green combs to ensure the correct spacers are used

Steps for 8.5% gel recipe

Summary

Protein gels are made up of two different gel layers; an upper and lower layer. The lower gel has a pH of 8.0 which helps separate proteins by molecular weight. The upper gel (which is prepared second) has a pH of 6.8 which helps focus proteins into a smaller volume.

Lower gel Recipe

• 5.6 ml of H20
• 2.8 ml of lower buffer (different in ion concentration and pH, contains SDS & tris, contains ultrapure water)
• 3.5 ml of 30% acrylamide (NOTE: acrylamide is toxic when it is aqueous but safe when polymerized, handle carefully
• ** kept in fridge** add when you are ready to fill glass plates, polymerization will start
• 100 ul of 10% radical APS (ammonium persulfate. To make: 1g of solid APS+ 10 ml DI water dissolved in a 10 ml tube)
• 10 ul TEMED

Steps for adding Lower Gel solution

1. Create the gel solution in a separate 50 ml falcon tube (create enough for the amount of gels you are creating)
2. Mix all of the ingredients together (adding APS and TEMED last) make sure you mix the solution before adding to glass plates
3. Use a dropper to quickly add the gel solution
4. Add a layer of DI water on top of the solution just added to keep gel fresh?
5. Use any remaining solution in the falcon tube as an indicator to when gel has polymerized (10-15 min)

If nothing polymerized after the time minimum time passed it could be due to no acrylamide added or bad TEMED/ APS

Upper gel Recipe

• 2.0 ml of H20
• 900 ul of lower buffer (different in ion concentration and pH, contains SDS & tris, contains ultrapure water)
• 600 ul of 30% acrylamide (NOTE: acrylamide is toxic when it is aqueous but safe when polymerized, handle carefully
• ** kept in fridge** add when you are ready to fill glass plates, polymerization will start
• 30 ul of 10% radical APS (ammonium persulfate. To make: 1g of solid APS+ 10 ml DI water dissolved in a 10 ml tube)
• 5 ul TEMED

Steps for adding Upper Gel solution

1. Once the lower gel has polymerized, remove the water layer last added (use a kimwipe)
2. Create the Upper gel recipe, and place the green 1.5 comb in carefully leaving a small layer of solution between the comb and lower gel
3. use a 1000ul pipette to quickly add the solution (just like the lower gel) in between one on the teeth of the comb
4. Use any remaining solution in the falcon tube as an indicator for when the gel as polymerized
5. Once the upper gel has polymerized, remove the combs, and your gel is ready to be loaded

Loading/ Running/ Transferring a Protein Gel

Loading a Protein Gel

(Identify how much protein you will have to load in the wells (excel sheet))

1. Take the gel apparatus and place it onto the running adapter (positive and negative screws) make sure knobs from apparatus are facing out
2. Place the adapter into the running chamber and fill the inner chamber with running buffer

Running buffer 1x solution: 100 ml running buffer (on bench) + 900 ml DI water

1. Fill the outside of the chamber with running buffer (ensure there is two seperate areas of liquid)
2. Add 4 ul of loading buffer to 1.7 ml tubes, amount of tubes depends on how many samples you have
3. Place protein samples on ice (remember to keep them on ice the whole time)
4. Using the Precision plus (blue) ladder [kept in fridge door], pipette 3.6 ul into the first well on both gels, keep on ice
5. Vortex tube, pipette amount of protein needed into 1.7 ml tube containing loading buffer
6. Take up the liquid from the tube (sample + loading buffer) and carefully pipette this mix into the well next to the ladder.

To load: press pipette towards you against the larger glass and carefully go between the plates into the running buffer. Slowly dispense all of the liquid (should fall to the bottom of the well) and remove the tip. Pull any bubbles from the pipette up towards the surface so it does not disturb your sample

1. Repeat 7 & 8 until all protein samples are added
2. Fill any remaining lanes with loading buffer

Running a Protein Gel

1. Connect (green) power top onto the running adapter. Ensure the screws are on tight and top matches with screws: black to black, red to red.
2. Set power supply to 100 volts for 1 hour and 30 min or until blue dye is running out of the gel
3. Switch on the power supply. Ensure it is running by visible bubbles rising inside