1. The optimum wavelength of light need for a spectrophotometric assay is determined by doing an absorbance spectrum
where the dependent variable is absorbance
and the independent variable is wavelength
.
2. What piece of equipment will you use in today's lab to help you determine the amount of product formed? Spectrophotometer
3. What dye was used in the first week's lab in order to determine the protein concentration of a sample? Coomassie Blue
4. A sample is put into a spectrophotometer. The intensity of light hitting the sample is 1350 watts. The intensity of light leaving the sample is 700 watts. What is the % transmittance?
Abs = log (Io/I) Abs = log (1/T) log (Io/I) = log (1/T) T = I/Io = 700/1350 x 100= 51.8%
5. In the first lab, you measured the amount of one type of macromolecule, protein. Briefly describe how you could measure the amount of a different macromolecule, DNA, present in a sample. Make a serial dilution of a known DNA. Use this to create a standard curve. Use the standard curve to determine your unkown. The procedure for detecting the DNA should be specific for DNA (a sort of Bradford assay for DNA).
6. A sample shows an absorbance of 0.85. Calculate the % transmittance.
Abs=log(1/T)
10^ABS=1/T
T=1/10^ABS=1/10^0.85
T=0.14
14%
7. What is the molarity of a solution that has 250 ug of the enzyme catalase in 150 ml water if catalase has MW=42,000?
(1/150 ml) x (10^3 ml/L) x (mole/42,000 g) x (1g/10^6 ug) x 250 ug =
3.96 x10^-8 M
8. What is the molar extinction coefficient of NAD if the solution has an absorbance of 0.52 in a 1.5 cm tube with a concentration of 40 uM?
Abs = E * C * X
Abs= 0.52, C= 40 uM= 40 x 10^-6 M, X= 1.5 cm
E= Abs/(CX)
=0.52/(40 x 10^-6)(1.5 cm)
= 8666.7 L/mol cm
9. The following serial dilution was done. Calculate the
concentration of the initial tube.
final
conc=1.5 M
total dilution= 1/5 x 9/10 x 8/10=36/250
DF=250/36
Ci=Cf x DF
Ci=1.5 x 250/36
=10.4 mM
10. You have 15 ml of an 55 mM solution of hemoglobin. How much of this hemoglobin should you transfer to get 8 ml of a 25 mM solution?
C1V1=C2V2
(55mM)x=(25mM)(8ml)
x = 3.63 ml