Swartz:Protocols/Protein gels

https://docs.google.com/document/d/11R2lvr_NIa2uSuxqNjvPmfPURieiF_ML3YOcAJmnA1M/edit

Running a Protein Gel

Protocol

Short Version

1. Withdraw 5l samples from the labeled protein synthesis reaction
2. Mix the samples with same volume of 2x PAGE sample buffer
3. Boil the mixture for 2 minutes
4. Load the sample on pre-cast SDS-PAGE gel
5. Run the gel electrophoresis
6. Dry the gel using a gel dryer
7. Expose X-ray film to the dried gel overnight
8. Develop the film

Long Version

1. 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

1. Incubate the samples 10 min at 70C

1. Prepare the gel
   1. Remove it from its wrapper
   2. Rinse it off with DI water
   3. Remove the tape from the slot
   4. Remove the comb
   5. Squirt DI water into the wells and shake it out
   6. 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