- The centrifugal force, gmax can be obtained from the following formula:
gmax = 1.12 r (RPM/1000)2 where r = radius of rotation in mm, RPM = revolutions per minute.
- Tabulate the activities of succinate dehydrogenase (SDH) in the 5 fractions, expressing the activity as change of
absorbance per minute, ΔA/min. Provide a descriptive legend for the table.
Table 1 ……………
| Cell Fraction | Absorbance (λ =600 nm) at start | Absorbance (λ =600 nm) after 1 mins | ΔA | ΔA/min* |
| 1 Brei | 0.981 | 0.942 | ||
| 2 Nuclear pellet | 0.847 | 0.761 | ||
| 3 Mitochondrial pellet | 0.521 | 0.474 | ||
| 4 Microsomal pellet | 0.509 | 0.468 | ||
| 5 Cytoplasmic fraction | 0.320 | 0.122 |
*These values can be thought of as being volume activities as they are all associated with 50 μL samples
4) Record the absorbance values from your protein standard solutions and your five fractions to complete Table 2. Provide a descriptive legend for Table 2.
Table 2 …………
| BSA µg | Absorbance 1 (λ =595 nm) | Absorbance 2
(λ =595 nm) |
| 0 | 0 | 0 |
| 5 | 0.177 | 0.383 |
| 10 | 0.589 | 0.356 |
| 15 | 0.376 | 0.431 |
| 50 | 0.456 | 0.444 |
5). Plot a protein standard curve using both duplicate Absorbance values (not the mean) for your BSA standard and draw your line of best fit. NB the protein (X) axis will be in amount of BSA protein (µg, not concentration units).
You may either plot this by hand and scan the image for here OR plot the graph in Excel and copy and past the result in here.
Provide a figure legend for your figure.
Figure 1 ……………………….
- For the cellular fractions, complete the table below.
- Enter the Absorbance values from the Protein Assay for the Cellular fractions
- Calculate the mean absorbance values for each cellular fraction and add this information to the table.
- Provide a descriptive legend for Table 3
Table 3
| Cell fraction | Abs 1
(λ =600 nm) |
Abs 2
(λ =600 nm) |
Mean Abs
(λ =600 nm) |
| 1.Brei | 0.665 | 0.778 | |
| 2.Nuclei | 0.652 | 0.751 | |
| 3.Mitochondria | 0.625 | 0.581 | |
| 4.Microsomes | 0.623 | 0.567 | |
| 5. Cytoplasm | 0.614 | 0.402 |
- Complete Table 4 below. To do this use the mean Absorbance values determined for the cell fractions in Table 3, and then using the Protein assay calibration curve created in step 4 estimate the amount of protein in each of your 5 μL samples of the cell fractions 1 to 5 (column A).
You should then express this as mg/50μL (column B), as you used 50 µL of sample in your earlier succinate dehydrogenase enzyme assay. (Multiply the value by 10 to get the amount in 50 µL and then divide by 1000 to get the number of mg of protein)
Table 4
| Cellular Fraction | A
Protein µg/5 µL |
B
Protein mg/50µl |
| 1 Brei | ||
| 2 Nuclear pellet | ||
| 3 Mitochondrial pellet | ||
| 4 Microsomal pellet | ||
| 5 Cytoplasmic fraction |
- Now that you have determined the protein concentrations in the fractions (in mg/50µL), you can take advantage of this and express your earlier volume enzyme activities as specific activities (i.e. change in activity/mg protein)
It is obtained by dividing your volume activity by the amount of protein in the sample that you assayed in mg – you have the information for this in the table 4 above, column B.
(An example calculation is if ΔA/ min (in 50 µL) was 0.2 and the amount of protein (mg/50µl) was 0.1, then the specific activity is 0.2/0.1 = 2 ΔA/ min/mg.)
Complete the Table 5 below for the cell fractions, and also provide a descriptive legend for the table.
Table 5
| Cell Fraction | Specific Activity of succinate dehydrogenase
(ΔA/min/mg) |
| 1.Brei | |
| 2.Nuclear pellet | |
| 3.Mitochondrial pellet | |
| 4.Microsomal pellet | |
| 5. Cytoplasmic fraction |
9.
(i) In Excel, draw a bar chart of Specific Activity of succinate dehydrogenase (SH) enzyme against Cell Fraction to show the distribution of the SDH marker enzyme throughout the five fractions that you have prepared.
(ii) Paste the bar chart figure below and provide a figure legend.
Figure 2 ……………………….
END OF THE PROFORMA
