Difference between revisions of "SDS-page gels"

Latest revision as of 20:15, 23 July 2018

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

Running Gels in the Cold Room (optional)

• In the cold room

1. Make sure power source is off
2. Connect red to red and black to black
3. Turn the power source on
4. Switch to milliamps on the first switch
5. Switch volts to amps on the second switch
6. Set for 2 hrs, 220, hit “run”
7. Check that it is running (bubbles rising)
8. Check after 30 minutes that it is still running

Transferring a Protein Gel

1. Once run is complete (blue is running out of gel) switch off power supply
2. Place transfer buffer into a container (found on drying rack)

Transfer buffer: 28.8g glycine, 6.07g tris base, 200 ml methanol

1. Build your sandwich in transfer buffer (two sandwiches)

• White side of sandwich holder down
• One black mesh pad
• 2 layers of filter paper
• One piece of nitrocellulose membrane (not PVDF) fitted to gel size
• Your gel

To remove gel: take glass plates carefully out of the glass plate holder Remove the smaller glass plate by slowly turning one spacer towards you, lifting the plate and making the gel stick to the larger plate Take the larger plate into the solution with the sandwich and use a razor blade to carefully pry the gel from the larger glass plate Once it is onto the membrane in solution, adjust so gel is centered on membrane

• 2 layers of filter paper
• One black mesh pad

1. Black side of sandwich holder down, and close tight
2. Place both sandwich holders into transfer apparatus with both black sides facing the black wall of the transfer apparatus. Put the entire apparatus into the transfer chamber
3. Place an ice block (from the freezer) into the chamber and fill with transfer buffer to the top
4. Place the chamber top on, and switch on the power supply
5. Transfer the gel at 500 volts/ 200 milliamps on the bench, overnight