1.What was the purpose of measuring the stock solutionâs absorbance at different wavelengths? Or in other words, why did you need to make an absorbance spectrum for this dye?
2.Explain why you used a blank when using the spectrophotometer. Make certain to explain why you needed to use the blank between each measurement in the first part of the experiment, but only once in the second part of the experiment.
3.You mix up a stock solution of green dye by adding 5 mg of green dye in 100 ml of water and find that there is maximum absorbance of 0.75A at 425nm for this dye.
What wavelengths of light do you expect to be transmitted through this solution (refer to Table 3.1 in your lab manual)?
If you were to add another 2 mg of the green dye to your stock solution would you expect the absorbance at 425nm to be higher or lower than 0.75 A425?
1.What was the purpose of measuring the stock solutionâs absorbance at different wavelengths? Or in other words, why did you need to make an absorbance spectrum for this dye?
2.Explain why you used a blank when using the spectrophotometer. Make certain to explain why you needed to use the blank between each measurement in the first part of the experiment, but only once in the second part of the experiment.
3.You mix up a stock solution of green dye by adding 5 mg of green dye in 100 ml of water and find that there is maximum absorbance of 0.75A at 425nm for this dye.
What wavelengths of light do you expect to be transmitted through this solution (refer to Table 3.1 in your lab manual)?
If you were to add another 2 mg of the green dye to your stock solution would you expect the absorbance at 425nm to be higher or lower than 0.75 A425?
Related textbook solutions
Related questions
BIOCHEM LAB HELP:
here are our data :
| concentration | absorbance |
| 0.02mL | 0.071A |
| 0.04mL | 0.090A |
| 0.06mL | 0.115A |
| 0.1mL | 0.156A |
| 0.2mL | 0.282A |
| 0.3mL | 0.357A |
| 0.4mL | 0.423A |
| 0.6mL | 0.626A |
| 0.8mL | 0.811A |
| 1.0mL | 1.040A |
| UMKNOWN | 0.546 |
please answer all questions
Spectrophotometric Analysis of Protein
1. Provide the standard curve for your experiment and the calculation of the unknown concentration from absorbance.
2. Is the concentration of the unknown you calculated from your standard curve the same as the concentration of the unknown stock solution? Why or why not?
3. Explain the process through which the Biuret reagent complexes with proteins to produce the characteristic color.
4. Why is it important to condition your cuvette prior to each reading?
5. Why is it important to blank your spectrophotometer, prior to use?
6. The lab manual discusses how proteins have characteristic absorption at 275-280 nm due to the presence of tyrosine and tryptophan (to some extent phenylalanine as well) in addition to the absorbance that occurs at 190 nm. Why not simply measure the absorbance of our protein sample at this wavelength and forego the Biuret reagent entirely? Discuss this question in light of the information provided in your lab manual, your knowledge of protein structure, and limitations inherent to a teaching laboratory.
7. According to Beerâs law, the relationship between concentration and absorbance should be perfectly linear, as would be indicated by a linear regression (R^2) value of 1. However, often the regression value is less than one, indicating an imperfect correlation. Considering the possible limitations of the Beer-Lambert law, and give a detailed explanation as to why this result may have been observed.
yeast population dynamics
Procedure
1. Work in pairs on this lab, so 12 tubes per pair of students. And share a tube rack with one other pair of students
2. Turn on your spectrophotometer. It needs at least 15 minutes to warm up to give you good readings.
3. Add 5 mL of yeast extract solution (YECM) to each of 12 tubes. (The yeast extract provides vitamins and amino acids for yeast growth and will be the same for all cultures). The tubes should be labeled with your initials, treatment, and tube number. Tape or Parafilm down the lids of 3 tubes, and label them âCONTROLâ.
Do not touch the insides of the tubes or lids! Try to keep these as sterile as possible!!
4. Add 50 mL live yeast culture to each of the remaining 9 tubes.
5. Add the varying volumes of sugar and/or ethanol using Table 1 below.
6. Use Parafilm to close the tops of each tube, making sure the Parafilm is tight and no air can get in, and label each tube with the following:
Amount of sugar added (mL) Amount of ethanol added (mL)
Name of your group Tube number
| Table 1: setup yeast tubes (remember, 1 mL = 1000 mL) | ||||
| Tube number | Yeast culture medium? (5 mL) | Live yeast culture? (50 mL) | Sugar added (mL) | Ethanol added (mL) |
| 1 â control | YES | NO | 0 | 0 |
| 2 â control | YES | NO | 0 | 0 |
| 3 - control | YES | NO | 0 | 0 |
| 4 | YES | YES | 0 | 0 |
| 5 | YES | YES | 0.25 | 0 |
| 6 | YES | YES | 0.5 | 0 |
| 7 | YES | YES | 0 | 0.25 |
| 8 | YES | YES | 0.25 | 0.25 |
| 9 | YES | YES | 0.5 | 0.25 |
| 10 | YES | YES | 0 | 0.5 |
| 11 | YES | YES | 0.25 | 0.5 |
| 12 | YES | YES | 0.5 | 0.5 |
Procedure for measuring absorbance (in absorbance units, or AU)
7. Calibrate the spectrophotometer:
Turn on the spectrophotometer and let it warm up for 15 minutes. You will get erroneous results if you donât let it warm up first.
Be sure the spectrophotometer is set to read at the wavelength of 550 nm
With no tube in the spectrophotometer and the lid closed, use the left-hand knob to adjust the reading to 0% Transmittance/push zero button to calibrate
Insert a CONTROL tube (making sure it is clear, without bacterial contamination which would make it cloudy), and use the right-hand knob to readjust the spectrophotometer to 100% Transmittance.
When reading the absorbance, be sure to line up the needle on the spec with its reflection.
8. Immediately before reading any tube, vortex the tube so that the spinning column reaches the bottom of the tube for several seconds. This is critical! The yeast cells are heavy and will tend to sink to the bottom of the tube, so you must vortex the tubes to resuspend them: otherwise, your spectrophotometer readings will be erroneously low. If the vortex is not enough to suspend the pellet of yeast cells at the base of the tube, take a piece of Parafilm and cover the top of the tube, then cover this with your thumb and shake the tube vigorously. The pellet should dislodge and the yeast cells should be easily resuspended after doing this. Use a Kimwipe to wipe down the outside of each tube, to remove fingerprints and other smudges that could affect the absorbance reading. (COULD BE A POTENTIAL ERROR)
9. Record the absorbance (in absorbance units, AU) for the tube on your data sheet.
10. Repeat steps 5 and 6 for every tube.
11. Leave the spectrophotometer turned on for the next user.
Figures you should include are:
Average absorbance vs. time for the no ethanol (0 mL) treatment
Average absorbance vs. time for the 0.25 mL ethanol treatment
Average absorbance vs. time for the 0.50 mL ethanol treatment
Sugar added vs. average carrying capacity (K). Use different symbols to denote each of the three alcohol concentrations
A. After a limit, the increasing concentration of sugar decreases the carrying capacity and growth rate. This is because at higher sugar concentrations, the medium becomes hypertonic and the yeast cells loss water towards the medium.With increasing concentration of the ethanol, the carrying capacity and the growth rate decreases. Why does this happen?
B. Is there any interaction between the effects of adding sugar and alcohol on yeast?
C. why do some cultures not reach K?
D. What are the potential sources of error and assumptions made in this experiment?
E. What do these results mean in a more general (non-yeast) context?
3. Equilibrium
Insulin resistance occurs when cells no longer bind insulin in a normal manner, thus inhibiting the cells from transporting glucose inside the cell. Without intercellular glucose the cells cannot produce energy to support life.
Letâs suppose part of your project requires you to study the equilibrium reaction between the new variation of insulin you synthesized and cell receptors on the walls of cells. This is the equilibrium expression you propose.
cell receptor + free insulin cell receptor-insulin complex
or in abbreviated terms
R + I R-I
You devise a method of measuring the free insulin in solution. You run two experiments under the same conditions, except one experiment uses your modified insulin and the other experiment uses normal insulin. Table 1 below provides the initial conditions of your experiment and Table 2 provides the data you collected after equilibrium was established.
Initial Conditions for both experiments â all environmental conditions are kept constant (e.g. temperature at 34oC, pressure at 1 atm, pH at 7.4). A single stock nutrient and cell receptor solution was prepared with a concentration of 70 uM. 50 mL the solution was used in all experiments. The insulins were added as powders, so the volumes essentially remained unchanged.
Table 1: Initial Experimental Conditions
| Experiment 1 | Experiment Control |
| [free modified insulin] = 100.0 uM | [free normal insulin] = 100.0 uM |
| [cell receptor] = 70.0 uM | [cell receptor] = 70.0 uM |
Table 2: Unbound Free Modified Insulin and Unbound Free Normal Insulin Levels at Equilibrium
| Experiment 1 | Experimental Control |
| [free modified insulin] = 35.0 uM | [free normal insulin] = 50.0 uM |
Write the equilibrium expression for this reaction using [I] to represent the insulin concentration, [R] the cell receptor concentration, and [I-R] to represent the cell receptor-insulin complex concentration. (10 pts) Show all work.
Determine an equilibrium constant for both the modified insulin and the normal insulin reaction at 34oC based on the data above. (10 pts)
| [free modified insulin] | [receptor] | [Insulin-receptor] | |
| I | |||
| C | |||
| E |
| [free normal insulin] | [receptor] | [Insulin-receptor] | |
| I | |||
| C | |||
| E |
Which type of insulin binds more strongly to the cell receptor? Explain your answer. (10 pts)
Suppose the concentration of the receptor in the stock solution dropped to 50.0 uM. If you ran the experiment again with the same initial concentration of free modified insulin as above, what would you expect the free modified insulinâs equilibrium concentration to be? (10 pts)
| [free modified insulin] | [receptor] | [Insulin-receptor] | |
| I | |||
| C | |||
| E |
Can someone please help me with this? I dont understand what to fill in the ice tables.
