Issel Lim
7.021 Results – Protein Biochemistry
Experiment I-A: Lysing the bacteria and precipitating with ammonium sulfate
Results
The bacterial strain of His461 was frozen in July of 2001, and the 7.5mL suspension
appeared opaquely beige. After thawing, it became a milky fluid, with a pungent rotting smell.
Addition of the clear lysozyme caused the bacterial suspension to become more viscous, with
beige chunks in the milky fluid. Cold ddH2O with 0.2% Triton X-100 made the suspension less
viscous, with white chunks and a snot-like consistency. Using a pipette to add MgCl2 and DNase
I caused the suspension to cling to the pipette as it became less chunky and more foamy.
We experienced a severe loss of fluid because the centrifuge tube was dropped on the
floor. The final volume of the suspension was approximately 1mL. The volume of the
supernatant after centrifugation was approximately 1200μL.
Table 1: Approximate volumes and appearances upon each addition to the His461 suspension
Approximate
Time of
Volume
addition
~7.5mL
1:45pm
Opaque, beige
~7.3mL
2:00pm
Milky fluid, rotting smell
Addition of lysozyme
~7.5mL
2:04pm
Addition of cold ddH2O w/
Triton X-100
Addition of MgCl2 and
DNase I
Final (after centrifugation)
~7.8mL
2:14pm
~10mL
2:26pm
~1.2mL*
3:15pm
More viscous, beige chunks in
milky fluid
Less viscous, white chunks, snotlike appearance
Sticky around pipette opening, more
foamy, less chunky
Beige pellet, translucent
supernatant
Original His461 frozen
suspension
After thawing
*after spillage of the centrifuge tube
Appearance of suspension
Table 2: Appearance of the crude-lysate components (after centrifugation):
Crude lysate component
Crude lysate (CL) (before centrifugation)
Crude lysate supernatant (CL-S)
Crude lysate pellet (CL-P)
Volume
1.2mL
1.1mL
0.2mL
Appearance
Turbid, cloudy whitish
Clear
Cloudy whitish
Calculation: Addition of (NH4)2SO4 to the crude-lysate supernatant from 0% to 45% saturation
of (NH4)2SO4:
(percentage) = (amount of (NH4)2SO4) / (total amount)
Exp. I-A
0.45 = [x (NH4)2SO4] / [1000μL + x]
x = 818.18μL of (NH4)2SO4 to add to 1000μL of solution
Experiment I-B: Quantitative Assay of β-galactosidase Activity
We measured the amount of β-galactosidase activity by reacting concentrations of β-gal
with ONPG and measuring the optical density at 420nm (OD420) to determine which sample
contained the most β-gal. After practicing with β-galactosidase standards, we then measured the
samples from the crude-lysate centrifugation.
Calculation: How to make different concentrations of β-gal standards
(amount needed of standard) * (concentration of standard)
= (concentration needed) * (amount of solution) * (1mL/1000 μL)
Exp. I-B:
β-gal concentration from 100μg/mL to 25μg/mL:
(x) (100μg/mL) = (25μg/mL) (20μL) (1mL/1000μL)
= 0.005mL
= 5μL of β-gal into 15μL of Z-buffer to get a 20μL solution
β-gal concentration from 100μg/mL to 6.25μg/mL
(x) (100μg/mL) = (6.25μg/mL) (20μL) (1mL/1000μL)
= 0.00125mL
= 1.25μL into 18.75μL of Z-buffer
*note: (25 - 12.25)/2 = 6.25 = 1:4 dilution of 25μg/mL
6.25μg/mL = (5μL of 25μg/mL solution) into 15μL of Z-buffer
Table 3: Spectrophotometric absorbance: OD420 readings for β-galactosidase standards
A420 for 25μg/mL
A420 for 6.25μg/mL
Time point
=500μL Na2CO3 + 100μL ONPG +
=500μL Na2CO3 + 100μL ONPG +
(5μL of 25μL/mL) + 500μL Z-buffer (5μL of 6.25μL/mL) + 500μL Z-buffer
t = 2 minutes
0.490 (very yellow)
0.101 (pale yellow)
t = 15 minutes
2.214 (inaccurate/out of range)
0.669 (more yellow)
4μL of each of the crude lysate samples were placed into 1mL of Z-buffer and reacted
with 100μL of ONPG.
Table 4: Spectrophotometric absorbance: OD420 readings for crude lysate samples
A420 for Crude Lysate
A420 for Crude Lysate
A420 for Crude Lysate
(CL)
Pellet (CL-P)
Supernatant (CL-S)
Time =4μL CL + 100μL
=(4μL CL-P) + 100μL
=(4μL CL-S) + 100μL ONPG
Point ONPG + 500μL Na2CO3 ONPG + 500μL Na2CO3 + + 500μL Na2CO3 + 500μL Z(min) + 500μL Z-buffer
500μL Z-buffer
buffer
t=2
t=15
0.068(inaccurate/out of
range)
0.389
0.004 (inaccurate/out of
range)
0.025 (inaccurate)
0.070 (inaccurate/out of
range)
0.404
Calculation: Total Activity for CL Samples
(absorption reading)*(dilution factor)/(#minutes in reaction mixture)* (1nmolONP/mL)/
(0.0045A420) * (#mL of reaction mixture) / (#enzyme sample analyzed) * (#mL in sample)
CL-S: @ t=15 timepoint
(0.404 A420*2)/15min * 1nmol ONP/mL/0.0045 A420/min * 1104μL/4μL * 1.2*10^3μL
= 3171U
CL: @ t=15 timepoint
(0.389 A420*2)/15min * 1nmol ONP/mL/0.0045 A420/min * 1104μL/4μL * 1.2*10^3μL
= 3053U
Because the range of accuracy on the spectrophotometer is 0.1 to 1.00 A420, CL-P was out
of range on both readings and hence no viable total activity could be calculated.
The crude lysate supernatant had the highest OD420 reading, and therefore contained the
most β-galactosidase.