Difference between revisions of "SDS-page gels"
From SDMRC
| Line 16: | Line 16: | ||
# 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 | # 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=== | ||
| + | # Create the gel solution in a separate 50 ml falcon tube (create enough for the amount of gels you are creating) | ||
| + | # Mix all of the ingredients together (adding APS and TEMED last) make sure you mix the solution before adding to glass plates | ||
| + | # Use a dropper to quickly add the gel solution | ||
| + | # Add a layer of DI water on top of the solution just added to keep gel fresh? | ||
| + | # 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 | ||
Revision as of 22:29, 17 July 2018
Contents
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
- Place the spacers onto the larger clean glass, by the edges, and place the smaller clean glass plate on top
- 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
- 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
- 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
- Create the gel solution in a separate 50 ml falcon tube (create enough for the amount of gels you are creating)
- Mix all of the ingredients together (adding APS and TEMED last) make sure you mix the solution before adding to glass plates
- Use a dropper to quickly add the gel solution
- Add a layer of DI water on top of the solution just added to keep gel fresh?
- 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
