Swartz:Protocols/Protein gels: Difference between revisions
(New page: https://docs.google.com/document/d/11R2lvr_NIa2uSuxqNjvPmfPURieiF_ML3YOcAJmnA1M/edit =Running a Protein Gel= ==Protocol== #setup the reaction mixture only 20ul is loaded into the wells,...) |
(No difference)
|
Revision as of 01:26, 28 June 2012
https://docs.google.com/document/d/11R2lvr_NIa2uSuxqNjvPmfPURieiF_ML3YOcAJmnA1M/edit
Running a Protein Gel
Protocol
- setup the reaction mixture
only 20ul is loaded into the wells, so I scaled down the reaction in the booklet
Sample: Enough for final protein conc > 0.03 mg/ml DI Water: To required volume 4X NuPAGE LDS 12.5ul DTT (0.5M) 5.0ul ------------ Total volume 50ul VORTEX
- Incubate the samples 10 min at 70C
- Prepare the gel
- Remove it from its wrapper
- Rinse it off with DI water
- Remove the tape from the slot
- Remove the comb
- Squirt DI water into the wells and shake it out
- If desired, the positions of the wells can be marked
4. Assemble the gel in the box a: Snap the white bracket into place The black wire goes on the right b: Put the flat, clear plastic slab behind the white bracket if only running one gel c: Put the gel in front of the white bracket The access to the wells is on the inside of the chamber created by the white bracket – The slot on the bottom of the gel is exposed to the outside d: Put the wedge without the screw in the back of the assembly e: Back the screw out of the other wedge f: Shove in this wedge so all of the pieces are really tight
5. Prepare Antioxidant solution a: 500ul is added to 200ml of the 1X NuPAGE running buffer & mixed b: Pour this solution into the center chamber created by the white bracket, gel, plastic slab c: Wait to be sure that none leaks into the outside chamber
6. Add NuPAGE running buffer to the outside chamber a: It need only go past the slot in the gel, halfway up b: Running buffer is by the power supplies, the concentrated stock is there too
7. Load the gel a: Pipette the antioxidant buffer into all of the wells b: SeeBlue ladder or MarkIV ladder can be added directly to the first well 20ul gives a dark ladder c: 20ul of the samples are added to the wells
8. Run the gel a: Program 8 can be used on the power supply 300V 60 mA (limiting) 110 W Approximately 1 hour b: Let the blue stain in the samples get about to the slot on the bottom.
9. Crack the gel open a: Cut off the thin well separators b: Cut the gel just above the slot on the bottom
10. Place the gel in a tray and cover with staining solution a: The staining solution is by the power supplies
11. Stain the gel There are two alternative methods a: Microwave 10s, swirl for 1 min Repeat for a total of 3X microwave b: Let the gel swirl in the stain overnight
12: Pour staining solution into radioactive stain waste a: located by the power supplies
13: Pour one cm of destaining solution on the gel a: located under the sink
14: Destain the gel There are two alternative methods a: Repeat the 10second microwave procedure 3X for destaining b: Destain by swirling in solution overnight The destaining solution should be changed at least once.
15: Dry and Mount the Gel a: Wash twice with DI water for 5 minutes b: Place in gel drying solution for 15 minutes Wet two pieces of celophane in this solution as well c: Mount the gel using the special square white tray Keep the surface wet to avoid air bubbles d: The solution can be recycled, next to the power supplies