Q1. Absorption spectrum for Lowry assay (10pt). Describe the absorption spectrum that you obtained in the Lowry assay, based on the one representative sketch that you made. (Optional: if it will help you with your discussion, you can include a photo of the sketch from your notebook, but this is not a requirement.) In your description, include the following:
Which sample was being assayed here (which tube number it was, and what sample that corresponded to).
The actual observed color of the sample (what you saw by eye).
The wavelength that was used in the calculations, and the wavelength that the instrument considered to be 'background' based on default settings for this procedure (see the Handbook, p.13).
Your assessment of whether those wavelengths used in the calculations (the one that indicates that protein is present, and the one that indicates only background - no protein) were appropriate. Did you indeed observe high and nearly-undetectable absorbance, respectively, at those two wavelengths when protein was present?
The range of wavelengths that you estimate to be detectable above the background. (In other words, what other wavelengths could the software have potentially used in order to perform the protein concentration calculations?)

Q2. Lowry raw data (5pt). Provide a clearly-organized table that contains all of the numerical data that you collected from the NanoDrop for the 12 tubes that were set up. If you have any missing data, clearly explain the reason.

Q3. Standard curve and calculation of unknowns in Lowry assay (10pt).
Provide the following:
a. A graph of the standard curve, with a regression line through the data and the r2 value indicated on the graph. (If you are unsure of how to do this, note that it will be performed in lab for certain kinetics graphs during Lab 4A, before the due date for this assignment question.)

b. A paragraph that discusses whether or not all three concentrations of your unknown (one undiluted and two diluted) fell within the range of absorbance readings over which the standard curve is valid.
A clear and specific statement of your best numerical estimate of what the actual concentration of the undiluted unknown was (remember to factor in that in some cases your unknown had been diluted!). Be sure to explain how you arrived at this estimate, making specific reference to your numerical data.

Q4.
Three recommendations for the future in Lowry assay (5pt). If you had to repeat this experiment, what three things would you focus on doing differently?
Focus on things that can be achieved in the time that we have available and using equipment that we already have.
You may have other ideas, but consider these options: out al you
Did you have enough time to incubate your mixtures for the recommended amounts of time? (If not, can you think of a way that you could have saved time in another part of the lab procedure?)
Do you think that you were able to get a reliable standard curve without performing replicate readings on any of your tubes?
Do you think that you did enough replication in order to get reliable TO readings for the unknowns?
Do you think that it would have been a better learning experience if the students had prepared all of the protein dilutions for the standard curve?
Did any of your spectra have unexpected shapes, and if so then can you think of any ways to address this in the future?

Q5. SDS-PAGE gel image (10pt). The response to this question should include two
components:
a. The image of your SDS-PAGE gel.
b. A paragraph describing what samples were run in the gel, and under what conditions the gel was run. This is not the results interpretation - it simply explains captions your image by explaining what type of gel was run, and what samples were run. Be sure to include the following: The % of polyacrylamide,
How long the gel was run for and at what voltage. Chemical contents of the sample loading buffer.
Which samples are found in which lanes, and the heat treatment that the samples received before being loaded.
The name and source of the molecular weight marker.

Q6. SDS-PAGE gel analysis (10pt). The response to this question should discuss each of the five sample types individually, separated into five different short paragraphs as follows:
a. BSA: did the sample migrate as expected? Make specific reference to the bands, as compared with the molecular size marker.
b. alkaline phosphatase: did the sample migrate as expected? Make specific reference to the bands, as compared with the molecular size marker.
c. chymotrypsin: did the sample migrate as expected? Make specific reference to the bands, as compared with the molecular size marker.
d. malate dehydrogenase: did the sample migrate as expected? Make specific reference to the bands, as compared with the molecular size marker.
e. Isolated DNA from Lab 1A: did the samples appear to contain any protein contamination? If so, estimate size(s).
f. If there were problems in the running of your SDS-PAGE gel, discuss them here in a final paragraph - what should be done differently in the future?

Q7. Protein concentration by A280 method (10pt). Based on the calculated protein concentration for each sample, and the volume that was loaded into the gel, indicate the calculated mass of each protein that was loaded into each gel well. Include the following three components:
a. Create a data table that includes the following columns, based on NanoDrop measurements (keep in mind that mg/ml and ug/ul are equivalent units!):
Sample name (give all six)
A280
own
A260/A280
Concentration (ug/ul)
Volume loaded on SDS-PAGE gel (ul; review the procedure and be sure that you are reporting only sample volume here, not sample + loading buffer!)
Mass loaded on SDS-PAGE gel (calculate this using other columns in this table; be sure to include units!)
b. Include a short paragraph about your judgment regarding whether the calculated mass of protein correlated with overall band brightness in each well. (e.g. if one type of sample stained darker than the rest, did you also see a higher calculated concentration for this sample, or not?)
C. Three methods have now been used to assess whether the two DNA isolation methods from Lab 1A allow protein contamination of the DNA samples (SDS-PAGE, Lowry assay, and A280 assay). Based on these three lines of evidence, make an overall concluding statement (or short paragraph) regarding the two methods with regards to whether they permit contaminating proteins into the final DNA sample. (Do both isolation methods show signs of protein contamination? Only one?
Neither? And do the methods of determining protein contamination agree with each other or not?)

Q1. Absorption spectrum for Lowry assay (10pt). Describe the absorp...