PROTOCOL FOR SDS-PAGE

Primary considerations:

• Volume of solution depends on number and thickness of gels:
  • Hoeffer 600 apparatus uses 18 x 16 cm gels
  • thickness can be 1.5 or 0.75 mm depending on spacers used
  • Lower (resolving) Gel requires:
    • 1.5 mm thickness: 30 ml
    • 0.75 mm thickness: 15 ml
  • Upper (stacking) Gel requires:
    • 1.5 mm thickness: 10 ml
    • 0.75 mm thickness: 5 ml
• Percent acrylamide depends on size of proteins to be resolved
  • 5% - IgM and high MW proteins (minimum usable percentage)
  • 7.5% - serum protein spectrum
  • 12.5% - Ig subunits

Basic Recipe Table: Lower (resolving) Gel

1 18x16 cm 1.5 mm gel (30 ml)

Calculate volumes required and fill in protocol steps below

Lower (Resolving) Gel Preparation

1. Add _________ ml RO H₂O into a 500 ml side-arm flask using 10 ml pipet with electric pipet filler.
    
2. Add _________ ml of 4x Lower Tris Buffer using the same pipet
   Stored on SDS-PAGE stock solution shelf.
   nbsp
3. Add _________ mL of 30/8% Acrylamide/Bis using a new 10 ml pipet
   Stored at 4C
   Dispose pipet in HAZARDOUS WASTE container.
    
4. Add _________ uL of 10% SDS using an automatic pipeter.
   Stored on SDS-PAGE stock solution shelf).
    
5. Add _________ uL of the APS solution using automatic pipeter.
   
6. Swirl gently to mix solution without introducing air.
   
7. Attach flask to vacuum line for 15 minutes to degas.
   Secure flask with clamp and use flat surface of oversized rubber stopper to seal flask.

Prepare Gel Plates in Casting Stand

1. Use gloves to prevent contaminating glass surfaces with hands
2. Place first glass plate on bench.
   Widest dimension is horizontal.
3. Inspect upper surface for lint and fingerprints.
   Clean if necessary.
4. Place spacers in position on top
5. Place second plate in position
   Add additional spacers and plates if dual gels are used.
6. Compress outermost plates while placing clamps on edges.
   Using spatula to align spacers to outermost position flush with clamps.
7. Compress outermost plates and move the assembly to vertical position
   Align glass plates and spacers so bottom and top edges are flush.
8. Tighten screws in increments to point where they slip on gloved hands.
   Do not tighten all at once.
9. Place the gray rubber gasket in the bottom of the casting stand, smooth side up.
10. Place assembled plates in the stand.
11. Insert cam levers and rotate to tighten plates into position.
12. Repeat the process for the other plate assembly.

After de-gassing monomer solution:

1. use extreme caution in breaking vacuum- detach vacuum hose slowly
    
2. Add _______ uL of TEMED using automatic pipter
   Stored at 4C.
3. Swirl gently to mix solution without introducing air.
    
4. Immediately transfer _________ ml of the solution between the plates using a 10 ml pipet and electric pipet filler.
5. Layer water-saturated isobutanol (top phase) onto the solution using a pasteur pipet.
6. Allow 60 min for complete polymerization.

Prepare Samples and Standards

1. Label sets of 1.5 ml microcentrifuge tubes
2. Add 100 ul of 10x Sample Buffer to each Sample tube using 100 ul Hamilton syringe
   Stored on SDS-PAGE Reagent shelf.
3. Wash syringe by repeated rinsing of RO H₂O
4. Add 10 ul of serum sample to each tube using 10 ul Hamilton syringe
5. Rinse syringe between samples with 1x Tris-Glycine Running Buffer
6. Add 10 ul of 10 g/l chicken IgG standard solution to a tube containing 100 ul of 10x Sample Buffer
   Stored at 4C
   An additional tube of IgG Standards may need to be prepared depending on the number of gels.
7. Heat Samples and Standards in Sample Buffer on 100C heating block for 4 min
   Ensure that tube tops do not open during heating.
8. Allow to cool and quickly centrifuge to collect condensate

Prepare Upper (stacking) Gel

Basic Recipe Table- Upper (stacking) Gel

1 18x16 cm 1.5 mm gel (10 ml)

Calculate volumes required and fill in protocol steps below:

1. Add _________ ml RO H₂O into a 250 ml side-arm flask using 10 ml pipet with electric pipet filler.
    
2. Add _________ ml of 4x Upper Tris Buffer using the same pipet
   Stored on SDS-PAGE stock solution shelf.
    
3. Add _________ mL of 30/8% Acrylamide/Bis using a new 10 ml pipet
   Stored at 4C
   Dispose pipet in HAZARDOUS WASTE container.
    
4. Add _________ uL of 10% SDS using an automatic pipeter.
   Stored on SDS-PAGE stock solution shelf.
    
5. Add _________ uL of the APS solution using an automatic pipeter.
   
6. Swirl gently to mix solution without introducing air.
   
7. Attach flask to vacuum line for 15 minutes to degas.
   Secure flask with clamp and use flat surface of oversized rubber stopper to seal flask.

After the Lower Gel has polymerized for 60 min:

1. Invert casting stand over sink and rinse out isobutanol with RO H₂O.
    
2. Add ______ ul TEMED and swirl gently to avoid introducing air.
3. Immediately transfer the solution in between the glass plates.
4. Insert combs between the glass plates.
   Insert at an angle to prevent air bubbles from becoming trapped at the bottom of teeth.
5. Add as much remaining solution as possible to the top of the gel.
6. Allow gel to completely polymerize for 60 min.

Prepare Tris Running Buffer

1. Add 250 ml of 4x Tris Running Buffer to a 1000 ml graduated cylinder.
2. Add 10 ml. of 10% SDS to the cylinder using a 10 ml pipet.
3. Add RO H₂O to 1000 ml.
4. Pour this solution into the bottom reservoir and add magnetic stirrer.
5. Add 150 ml of 4x Tris Running Buffer to a 1000 ml graduated cylinder.
6. Add 6 ml. of 10% SDS to the cylinder using a 10 ml pipet.
7. Add RO H₂O to 600 ml. Set aside.

Load Samples

1. Move gel casting stand to sink
2. Pull the combs out- straight upwards.
3. Wash-out the wells three times with the 1x Upper Tris Buffer squeeze bottle.
   Fill the wells with this solution.
4. Add rubber gaskets to the Top Reservoir
   Notice ridges that sit in the recess.
5. Lower the Top Reservoir onto glass plates in casting stand.
6. Remove cam levers from lower slots and insert into upper slots and turn to tighten.
   Be sure cam levers are pushed all the way into slot before tightening.
7. Add 300 ml. Tris Running Buffer to Top Reservoir.
8. Tape the clear plastic sheet with well numbers in the corresponding position
9. Load samples according to the Gel Run Log using a 10 ul Hamilton syringe.
   Wash the syringe between samples with 1x Tris Running Buffer.
   To assist visualizing the wells and sample load, you can add a wisp of Tracking Dye using an automatic pipeter. Avoid adding too much tracking dye.
   1. Gel A:
      1. Load samples to Lanes 1-10
      2. Load IgG standards to Lanes 11-15 in the the following order: 10, 8, 6, 4 and 2 uL.
      3. Load samples to Lanes 16-28.
   2. Gel B:
      1. Load samples to Lanes 1-10
      2. Load 1 ug/ul IgG standards to Lanes 11-15 in the the following order: 2, 4, 6, 8 and 10 uL.
      3. Load samples to Lanes 16-28.
   3. Gel C:
      1. Load samples to Lanes 1-9
      2. Load 1 ug/ul IgG standards to Lanes 10-14 in the the following order: 10, 8, 6, 4 and 2 uL.
      3. Load samples to Lanes 15-28.
   4. Gel D:
      1. Load samples to Lanes 1-9
      2. Load 1 ug/ul IgG standards to Lanes 10 -14 in the the following order: 2, 4, 6, 8 and 10 uL.
      3. Load samples to Lanes 15-28.
10. Be sure to add comments for missed lanes or botched loadings in the Gel Run Log.

Final Assembly of Apparatus

1. Lower Top Reservoir around Cooling Unit.
   Electrical posts should be on the same side.
2. Disperse bubbles under gels using curved pipet.
3. Slowly add the remaining Upper Tris Buffer to the Top Reservoir.
   Minimize turbulence to prevent disrupting samples in wells.
   Note level of Tris Running Buffer in Top Reservoir.
4. Place on magnetic stirrer.

Run Gels

1. Attach lid and connect electrical leads.
2. Turn-on power supply with rocker switch on lower left side.
3. Push the Select Mode membrane switch to the toggle to the mA position.
4. Push the Display membrane switch to toggle to Set position.
5. Adjust to 17.5 mA/gel (1.5 mm) using the large knob.
6. Push the the On/Off membrane switch to start run.
7. Record the mA, V and time on the The Gel Run Log.

Post-Run Disassembly

1. Add 200 ml 25% Trichloroacetic Acid (TCA) to a 500 ml graduated cylinder.
2. Add 200 ml RO H₂O to the cylinder for 400 ml total.
3. Pour this solution into plastic staining tray and place next to sink.
4. Record final mA, V and time on the The Gel Run Log.
5. Turn off the power supply first with membrane switch then rocker switch.
6. Detach lid and electrical posts.
7. Slide Top Reservoir upward and move to sink.
8. Pour buffer in Top Reservoir into sink.
9. Lay assembly on its side
10. Remove cam levers on top gel set while holding glass plates.
11. Separate glass plates with plastic or wooden spatula.
    DO NOT USE METAL.
12. Observe which plate the gel is adhering to and move the glass plate so that the gel is on top.
13. Place the glass plate on a clean level surface
14. Slice off the Upper (stacking) Gel approximately 1-2 mm above the Lower (resolving) gel interface using a single-edged razor blade.
15. Touch a paper towel to the Upper (stacking) Gel in order to remove it.
    Discard in HAZARDOUS WASTE container.
16. Rinse lightly with a stream of DI H₂O.
    Tilt the plate upward so that if the gel were to slide off, it would be blocked by your hand.
17. Invert the glass plate over the staining tray
18. Lower the glass plate into the solution and then lift so that the surface tension will peel the gel from the glass plate.
    Use a plastic or wooden spatula to loosen one cornor of the gel if necessary.
19. repeat the same procedure with the other gel(s).

Quantitative Staining

1. Add 0.5 g Coomassie Brilliant Blue G-250 to approx. 10 ml RO H₂O in a 15 ml centrifuge tube.
   Stored in Room Temperature Reagent Desiccator.
2. Vortex and place on rocking platform.
3. Add 400 ml of Stock Staining solution to 500 ml graduated cylinder.
4. Add 100 ml methanol to cylinder for a total of 500 ml. The solution will appear milky initially then turn to clear as the solution is mixed as it is poured into the staining tray.
   Methanol is TOXIC and is absorbed through unprotected skin.
   Use gloves and eye protection.
5. Incubate gel in TCA solution for 60 min on rocking platform.
6. Hold gels to bottom of staining tray with one hand and invert to discard TCA solution into sink.
7. Rinse with two changes of DI H₂O.
8. Add Stock-Staining-Solution/Methanol from cylinder.
9. Add Coomassie staining solution, which will include particulate material.
10. Stain 8 hrs (or overnight) on rocking table.
11. Add 100 ml of methanol to 500 ml graduated cylinder.
12. Add 300 ml of RO H₂O to cylinder to a total of 400 ml (25% methanol).
13. Remove staining solution by imversion
14. Wash any remaining stain particles off the sides of the staining tray with the methanol squeeze bottle.
15. Add 25% methanol de-staining solution.
16. De-stain for 4 hrs.
17. Replace with fresh 25% methanol solution and de-stained for an additional 4 hrs.
18. Remove de-staining solution, rinse gels twice with DI H₂O
19. Store in 25% ammonium sulfate solution.