Monday, January 24, 2011

Trypsin Digestion Protocol~

 Proteins are digested and chopped up into little pieces for further experiments.  In order to digest proteins, three solutions are used: DTT, IAA, and NH4HCO3.  A type of enzyme will be used to cut the protein at a specific amino acid.

PREPARATION FOR DIGESTION

1. DDT (Dithiothreitol) breaks up disulfide bonds formed by the bonds of cysteine. So, DTT is used to "unfold" the proteins and eliminate the bonds that make the protein folds.
  Prepare 1 ml of 100 ml of DTT
    molar weight: 154.25 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.01542 g =15.42 mg
2. IAA (Iodoacetamids) stabilizes the disulfide bonds broken up by DTT.  IAA prohibits the disulfide bonds to bond again.

  Prepare 1 ml of 100 ml of IAA
    molar weight: 184.96 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.018496 g =18.496 mg
3. NH4HCO3 is a buffer that stabilizes and helps the enzyme to rise to its maximum capability to clip and cut the protein.  A certain enzyme functions better with certain pH.  In this case, NH4HCO3 has a pH that works best with trypsin.
  Prepare 1 ml of 100 ml of NH4HCO3
    molar weight: 79.06 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.007906 g =7.906 mg

PROCESS OF PROTEIN DIGESTION
Two proteins used: BSA and apomyoglobin
1. BSA contains disulfide bonds; thus DTT and IAA are added to break the bonds.
  10 ul of BSA (3 ug/ul)given, add:
     5 ul DTT,
     25 ul NH4HCO3, and
     20 ul water.
     >incubate at 50 C for 45 minutes

    25 ul IAA, and
    25 ul NH4HCO3.
    >sit in darkness for 1 hour

The enzyme is then added according to a protein to enzyme ratio.  In this case, I used 50:1 (protein to enzyme in ug)
  BSA given: 10 ul of 3ug/ul
  50:1 = (3 ug/ul)*10 ul: x
  x= 0.6 ug
   trypsin stock concentration: 0.1 ug/ul
   0.1 ug/ul*x= 0.6 ug
   x=6 ul

   >add 6 ul of trypsin in the BSA solution and incubate at 37 C for 10 hours

2. Apomyoglobin only undergoes the protein digestion process
  Apomyoglobin given: 20 ul of 1 ug/ul, add:
    2 ul trypsin, and
    22 ul NH4HCO3.
   > incubate at 37 C for 10 hours


1 comment:

  1. Something wrong with the preparation for digestion:

    Prepare 1 ml of 100 ml of DTT etc makes no sense

    ReplyDelete