Electroelusion and dialysis

Electroelusion of proteins from stained gels 
  1.  Cut out stained protein spots on gel and soak in water.
  2.  Fill 750 ul of electroelusion buffer in the 2ml eppendorf tubes containing samples.
  3.  Shake for 30 minutes.
  4.  Cut 12 to 15 cm long seamless cellophane tubing small No 24.
  5.  Fill 250 ml MQ in 300 ml beaker, boil it for 5 minutes and keep the tubing in it.
  6.  Wet the small pieces of cellophane in a small beaker with water.
  7.  Open one end of the tubing membrane and close the other end by clipping.
  8.  Close the small end of the cup by wet cellophane and ring it.
  9.  Mount the other end with cellular tubing and ring it to stop the leakage.
 10. Fix the cup with the apparatus.
 11. Fill the cup toward the small end with samples.
 12. Fill the other end with elusion buffer in such a way that a layer of buffer should join 
     both the cup enabling the movement of protein from small cup to the cellular tubing.
 13. Fill the apparatus with electro elusion buffer.
 14. Sample should be toward the positive side.
 15. Run at 100 V for 2 hrs.
 16. Remove cellophane tubing and clip to close the end.
 17. Dialyse in cold room (4 ºC)
 18. Change the water for three time in 1st day.
 19. Next day change the water for two times.
 20. Transfer the proteinin to 2ml eppendorf tube.
 21. Freeze dry over night.
 22. Dissolve the protein in 30 μl of SDS sample buffer in two aliqute of 20 μl and 10 μl.
 23. Prepare 17% SDS-PAGE mini gel.
 24. Add 1ul sample buffer with 9 μl of SDS sample buffer and load this 10 μl.
 25. Load 2 μl low molecular weight marker. 
 26. Run it at 20- 25 mA 
 27. Silver stain to check the protein concentration

Silver Staining (Nakarai Kit)
  1. Shake the gel in solution 1 for 2 -5 hrs
  2. Replace with solution 2 and shake for 10 minutes and repeat this step for two times
  3. Relace with solution 3 and shake for 5 minutes three time.
  4. Wash with MQ for 5 minutes 3x
  5. Incubate in solution 4 shaking for 5-15 minutes
  6. Wash the gel with MQ water for 5 minutes 2x.
  7. Develop for 3 minutes in developing solution. 
      Note. Staining solution is considered as hazardous waste and should not be poured down the sink. 
      Place the staining solution in a disposable plastic container or hazardous waste container.
  8. Stop the reaction with acetic acid and wash with MQ water and air dry.

  Solutions needed for Silver staining

Solutions Chemical needed For 1 gel (ml) For 2 gels (ml) For 3 gels (ml)
Solution 1 Ethanol
Acetic Acid
Water
50
10
40
100
20
80
150
30
120
Solution 2 Ethanol
Acetic Acid
Water
10
5
85
20
10
170
30
15
255
Solution 3 Oxidizer or Synthesizer (kit)
Water
10
90
20
180
40
360
Solution 4 Solution A (kit)
Solution B (kit)
10
10
20
20
40
40
Solution 5 Developer (kit)
Water
5
95
10
190
20
380