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  1. May 2019
    1. Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA
    2. Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised
    3. Destaining of gel, trypsinization and peptide extraction
    4. 0.5-1 μg of DNA was used for each restriction enzyme digestion. 2-4 units of the restriction enzymes with the appropriate 10X buffers supplied by the manufacturers were used in a total reaction volume of 20 μl. The digestion was allowed to proceed for 6 h or 10min. (for FAST digest enzymes) at the temperature recommended by the manufacturer. The DNA fragments were visualized by ethidium bromide staining following electrophoresis on 1-1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments
    5. Restriction enzyme digestion and analysis