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.


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

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

Sunday, January 16, 2011

Bradford Protein Assay~

Happy LATE New Years~ I am sorry, I been busy studying for school (finals is coming up) and organizing data from the lab into my notebook. However.....

Recently, in my lab at CalTech, I did a lab on Bradford protein assay.  It is a procedure based on an absorbance shift of the Coomassie Brilliant Blue G250 dye.  When the dye binds with proteins, it convert from its original red form into a bluer form.  The bound form of the dye has an absorption spectrum maximum held at 595 nm.  Certain concentration of detergents, which is used to lyse cells, interferes with the Bradford assay.

I used Bovine Serum Albumin (BSA) as a known protein to create a calibration graph of concentration versus absorbance.

Prepare BSA solutions:

I was given BSA solution of 1 ug/ml, and I need to prepare BSA concentrates at 2 ug/ml, 5 ug/ml, 7 ug/ml, 10 ug/ml, 20 ug/ml, and 30 ug/ml.

Concentration (ug/ml)
BSA solution (based on 1 ug/ml)
Bradford reagent dye
2 ul
998 ul
5 ul
995 ul
7 ul
993 ul
10 ul
990 ul
20 ul
980 ul
30 ul
970 ul

All of the solutions have the final volume of 1000 ul or 1 ml.

I, then, input 2 ul of the solution into the nanometer, which has the Bradford protein assay procedure pre installed into the computer.

^ Instrument I used.

^The display.

A calibration graph is formed on the concentration versus absorbance.  The graph uses the Beer-Lambert Law:
(where Eb are constant, sometimes written as k and c as the concentration)

The calibration graph look something like this:

Notice the r squared variable.  In statistics, it is known as the coefficient of determination.  It determines the linearity of the graph.  In other words, the closer the variable is to 1, the more linear it is.  

Using the calibration graph, I was able to determine the concentration of several biological samples based on the absorbance of the samples and the corresponding absorbance over concentration.  

The following are a list of biological samples, which I performed the Bradford assay on in order to find the concentration:

Possible sources of error:
I performed the calibration graph and the actual experiment on two different days, so the results are not as accurate as they possibly are.
There are some samples with 0 concentration.  One thing is that I did not sanitize the nanometer correctly or air went into the machine while the experiment is performed.

This conclude the experiment. XD