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Proteomics/Experimental Protocols/Plant Proteomics about Two Dimensional Gel Electrophoresis

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Two Dimensional Gel Electrophoresis 1. Taking 4 tubes then one side covered with paraflim. 2. Prepare Straw Gel Buffer 3. In before Ampholine shacking by hand until this dissolve 4. Mixed through syringe 5. Straw Gel Buffer poured into the glass tube by syringe 6. Keep in minimum 6 hrs 7. Plant Sample preparation • Take the mature seeds • Poured the liquid N2 • Grinding with mortar with pestle • Lysis Buffer added in the sample before use lysis buffer are heated in wise bath at 1000 C for 3 minutes • Sample are placed in ice and mixed through the Vibra Cells • Spin down • Centrifuge at 70 C for 20 minutes • Take Albumin and Albumin Standard (DDW:Bradford=4:1) i.e.,(8000 +2000)µl • To prepare the 2 set sample and 1 set in 5 µl for UV-spectrophotometer, another for 2-DE • To make 3 standard sample (5, 10, 15 µl) • Another is placed in wise bath at 1000 C for 3 minutes. • To test the concentration through UV-spectra 8. To prepare vials and taking 10 ml sample buffer solution in vials 9. To check out the gel and placed in vials by using syringe. 10. To place in twist shaker for 1 hrs and 30 minutes 11. To remove the solution and again adding 10 µl buffer solution twice. 12. To prepare Separating Gel and poured and wait for 40 minutes. 13. To prepare Stacking gel and wait for 3-4 hours. 14. To run the machine into top portion (H3PO4) and lower portion (NaOH) 15. Sample are pour into the tube before remove the water 16. To start the machine(1st hr=200 V, 2nd hr=400 V and next 16 hrs=600 V)

17. To place the 1-D Gel on the 2-D Plate 18. To prepare Agarose gel 19. To place the marker and agarose gel. 20. To run the machine (80 mA for 1 machine, 160 mA for 2 machine) until end. 21. To separate the gel and soaking in Coomassie Blue before make Coomassie Blue. 22. To keep in min. 12 hrs.

Silver Staining Protocol 1. Fixing solution 40% Ethanol :

  1. Ethanol 100 ml
  2. Glacial acetic acid 25 ml
  3. Water 250 ml

NB : Soak the gel in fixing solution for 30 min. 2. Sensitizing solution :

  1. Ethanol 75 ml
  2. Sodium thiosulphate ( 5% w/v) 10 ml
  3. Sodium acetate (17 g) 1 packet
  4. Water 250 ml

Before use: Add 1.25 ml glutardialdehyde (25% w/v) NB : Remove the solution. Add sensitizing solution and leave shaking for at least 30 min.

  1. Washing 5 min.

3. Silver solution :

  1. Silver nitrate solution (2.5 % w/v) 25 ml
  2. Water 250 ml

Before use: Add 0.1 ml formaldehyde (37 % w/v) NB : Shaking 20 min. and washing 1 min 2 times. 4. Developing solution :

  1. Sodium carbonate (6.25 g) 1 packet
  2. Water 250 ml
  3. Stir vigorously to dissolve the sodium carbonate

NB : Shaking 2-5 min. 5. Stop solution:

  1. EDTA-Na¬¬2 .2 H2 O ( 3.65 g) I packet
  2. Water 250 ml

NB : Shaking 10 min. and washing 5 min in 3 times. 6. Water solution :

  1. Double distilled Water

In Gel Digestion

1. 200 µl DDW was placed in 500 µl tube. 2. Separate the gels from gel plate using 1ml blue tip. Slice the gels pieces, vortex 5 minutes and remove water. 3. Add 100 µl 25mM ABC/50% ACN, then vortex 10 minutes, spin down and remove the solution. Repeat 4-5 times.

    1. Overtaxing until to be white color for 30 minutes.

4. Placed in the speed vacuum 5. Adding 50 µl mM DTT/25 mM and then heating in wise bath at 560C for 45 minutes, then spin down and remove the solution. 6. Adding 50 µl mM IAA/0.1 % ABC and placed in dark place for 30 minutes. 7. No Spin down, remove the solution 8. Adding 50 µl 0.1M ABC, vortexing for 5 minutes and remove the solution 9. Adding 50 µl 100% ACN, vortexing for 15 minutes and removes the solution 10. Placed in the speed vacuum for 30 mins. 11. Adding 25 µl Trypsin (0.1 µg/µl), keep in refrigerator for 45 minutes at 40C 12. Adding 35-40 µl 25mM ABC (pH 8.0), incubate overnight at 370C in wise bath, then spin down, extract the trypsin. 13. To make ACN/DDW/TFA (660:330:10) µl 14. Adding 50 µl (ACN/DDW/TFA) in sample, keep in 30 minutes and overtaxing, repeat this step, spin down, and extract the solution and keep in solution. Repeat this step. 15. Placed in speed vacuum for 2 hours until 10-20 µl samples.

MALDI-TOF/MS Spectrophotometer:

1. Adding 5% Formic Acid in Gel Pieces (Coomassie Blue for 10 µl and Silver staining for 5 µl) and then pippetting. 2. Vortexing and Spin down 3. To make a solution A: 0.1% TFA B: 70% ACN,0.03% TFA Matrix Solution: (0.1% TFA 500µl + 50% ACN 500µl + CHCA 10 mg) 4. Zip Tip by B: 70% ACN,0.03% TFA ( 5~10 times) using C18 tips. 5. Zip Tip by A: 0.1% TFA ( 5~10 times) using C18 tips. 6. Sample in Zip Tip up & down (10 times) 7. A: 0.1% TFA Desalting 8. Matrix Solution(5-10µl) are placed in MALDI Plate 9. After drying, adding A: 0.1% TFA and remove 10. Adding DDW and remove

Submitted by Abu Hena Mostafa Kamal,Ph D student, Lab of Plant Proteomics,Dept. of Crop Science,College of Agriculture,Chungbuk National University,Cheoung-ju,361-763,south Korea

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