In-gel digestion protocol

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In-gel digestion Protocol
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1. Digest 3 BSA gel plugs and analyze with Ultraflex TOF/TOF with correct identifcation for all three. Repeat if fail.

2. Digest one BSA gel plug as control with the digestion of samples.

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Protocol
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1. Place gel plug in a 96 well plate.

•    1 pc/well for 2D gel-plug;
•    5 pcs of 1x1x1 mm3/well for coomassie;
•    10 pcs 1x1x1 mm3/well for silver stained.

2. Add 200 uL of sterile water shake for 5 min at room temperature.

3. Remove water and add 200 uL of 100% ACN. Shake for 5 min at room temperature.

4. Remove ACN and allow the gel plug to dry at room temperature for 5-10 min.

5. Add 50 uL 10mM dithiothreitol/10mM ammonium bicarbonate/well. Allow to sit at room temperature for reduction of disulfide bond for 1 hr.

6. Remove 10mM Dithiothreitol/10mM ammonium bicarbonate.

7. Add 50 uL 100mM Iodoacetamide/10mM ammonium bicarbonate. Keep in the dark place at room temperature for 1 hr.

8. Remove 100 mM Iodoacetamide/10mM ammonium bicarbonate.

9. Add 200 uL of 100% ACN. Shake at room temperature for 5 min. Remove ACN. Repeate this step twice. (Total 3 times). Repeat once more if the gel has not shrunk.

10. Add 20uL of 10ng Trypsin in 50% ACN/10mM ammonium bicarbonate (for coomassie, use 40uL). Leave at room temperature for 20 min before adding 30 uL of 30% ACN and leave at room temperature for 3 hrs.

11. Transfer liquid to another 96 well plate. (If the well is dry, add 30 uL of 30% ACN, shake at RT before transfering.

12. Extract the rest of peptide in 11 by with 30 uL 50% ACN/0.1% FA, and shake for at RT for 10 min. Transfer to the same plate in 10. Repeat twice.

13. Dry in a 40 degree incubator for 3-4 hr or overnight. A heat block or speed vac can be used.

14. Keep sample at -80C until analysis.

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Reconsitute sample in 5uL of 0.1 % FA.
Spot on MALDI plate with mixing ratio 1:1.
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Preparation of Reagents
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20 mM ammonium biocarbonate (FW 79.06) 50 mL.
Weigh 79.06 mg of ammonium bicarbonate. Dissolve in milliQ water adjust volume to 50 mL.

10 mM ammonium bicarbonate 50 mL
Add 25 mL of 20 mM NH4HCOO to a 50 mL volumetric flask. Adjust volume with water to 50 mL.

10 mM dithiothreitol/10mM ammonium bicarbonate 2 mL (freshly prepare).
Weigh DTT (FW 154.25) 3.085 mg dissolve in 10 mM ammonium bicarbonate adjust volume to 2 mL.

100 mM Iodoacetamide / 10 mM ammonium bicarbonate 2mL.
Weight Iodoacetamide (FW 184) 36.8 mg dissolve in 10 mM ammonium bicarbonate.

50% acetonitrile/10 mM ammonium bicarbonate
Add 100% ACN to 20mM ammonium bicarbonate 1:1

10ng trypsin in 50% ACN/10mM ammonium bicarbonate (for silverstain).
Add 2 mL of 50% ACN/10mM ammonium bicarbonate to 20ug of trypsin.

30% ACN 5mL (used 1.92 mL for 96 well).
Add 1.5 mL of 100% ACN to 3.5 mL milliQ water.

0.1% FA 50 mL
Add 50 uL of concentrate FA to 49.95 mL of milliQ water.

50% ACN in 0.1% FA 10 mL (8.64 mL for sample 96 well)
Add 100% ACN 5 mL in 0.1%FA mL.