# Difference between revisions of "20.109(S14):Protein-level and wrap-up analysis (Day7)"

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===Part 1: ELISA analysis=== | ===Part 1: ELISA analysis=== | ||

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#Open the text file containing your raw data in Excel, and save it as an Excel file. | #Open the text file containing your raw data in Excel, and save it as an Excel file. | ||

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#Average your replicate values for both standards and experimental samples. | #Average your replicate values for both standards and experimental samples. | ||

#Now calculate the average of your blank samples, then subtract this background value from each of your raw averages. (So far your column headings might look like: REP1, REP2, AVE, AVE-SUB.) | #Now calculate the average of your blank samples, then subtract this background value from each of your raw averages. (So far your column headings might look like: REP1, REP2, AVE, AVE-SUB.) | ||

+ | #*Note that putting dollar signs on either side of an Excel cell (e.g., $G$3) will allow you to point back to the identical value each time when you copy-paste, instead of moving down the row or column. And just for future reference, having a dollar sign on just one side or the other will keep column consistent but shift row, or vice-versa. | ||

#You will use your standard readings to make a calibration curve. Plot the absorbance readings for the standards (on the ''x''-axis) vs. the known concentration of collagen added (on the ''y''-axis). Just type in the first concentration, and divide by two down the column. | #You will use your standard readings to make a calibration curve. Plot the absorbance readings for the standards (on the ''x''-axis) vs. the known concentration of collagen added (on the ''y''-axis). Just type in the first concentration, and divide by two down the column. | ||

#Click in the chart area, then choose menu ''Chart'' → ''Add Trendline''. Click on the ''Options'' Tab, and choose to display both the equation and the R<sup>2</sup> value on the chart. | #Click in the chart area, then choose menu ''Chart'' → ''Add Trendline''. Click on the ''Options'' Tab, and choose to display both the equation and the R<sup>2</sup> value on the chart. | ||

#Delete data points that seem to be outside the linear range of the assay (just delete the AVE-SUB value, not the raw data!), until you get a reasonable R<sup>2</sup> value for your line, i.e., one that is close to 1. The equation should update in real-time as you delete data. | #Delete data points that seem to be outside the linear range of the assay (just delete the AVE-SUB value, not the raw data!), until you get a reasonable R<sup>2</sup> value for your line, i.e., one that is close to 1. The equation should update in real-time as you delete data. | ||

− | #Now that you have the slope and intercept of the line, you can feed this information back into the absorbance values for your experimental samples, and calculate the actual protein concentrations. If you are unsure of how to proceed, ask your peers or instructors. | + | #Now that you have the slope and intercept of the line, you can feed this information back into the absorbance values for your experimental samples, and calculate the actual protein concentrations. If you are unsure of how to proceed, ask your peers or instructors. |

+ | #*Again the $ symbol in Excel is useful here for efficient calculations, in case you want to compare the results for multiple slope/intercept values. | ||

#Your results will most likely be closer to ng/mL than μg/mL, so go ahead and convert them. | #Your results will most likely be closer to ng/mL than μg/mL, so go ahead and convert them. | ||

#Finally, address the following in your report: | #Finally, address the following in your report: |

## Revision as of 12:23, 6 May 2014

## Contents

## Introduction

Today we’ll complete the final piece of analysis for module 3, namely quantifying the collagen protein content in samples cultured under different conditions. You should spend the remainder of your time today finishing your module 3 report and preparing for your research proposal presentation.

## Protocols

### Part 1: ELISA analysis

- Open the text file containing your raw data in Excel, and save it as an Excel file.
- Label the columns to reflect your samples. It may be easiest to visually separate samples (in the first few rows of the sheet) and standards (in the next few rows), since they will undergo partly different manipulations. You may also want to work on two separate worksheets, one for each collagen type.
- Average your replicate values for both standards and experimental samples.
- Now calculate the average of your blank samples, then subtract this background value from each of your raw averages. (So far your column headings might look like: REP1, REP2, AVE, AVE-SUB.)
- Note that putting dollar signs on either side of an Excel cell (e.g., $G$3) will allow you to point back to the identical value each time when you copy-paste, instead of moving down the row or column. And just for future reference, having a dollar sign on just one side or the other will keep column consistent but shift row, or vice-versa.

- You will use your standard readings to make a calibration curve. Plot the absorbance readings for the standards (on the
*x*-axis) vs. the known concentration of collagen added (on the*y*-axis). Just type in the first concentration, and divide by two down the column. - Click in the chart area, then choose menu
*Chart*→*Add Trendline*. Click on the*Options*Tab, and choose to display both the equation and the R^{2}value on the chart. - Delete data points that seem to be outside the linear range of the assay (just delete the AVE-SUB value, not the raw data!), until you get a reasonable R
^{2}value for your line, i.e., one that is close to 1. The equation should update in real-time as you delete data. - Now that you have the slope and intercept of the line, you can feed this information back into the absorbance values for your experimental samples, and calculate the actual protein concentrations. If you are unsure of how to proceed, ask your peers or instructors.
- Again the $ symbol in Excel is useful here for efficient calculations, in case you want to compare the results for multiple slope/intercept values.

- Your results will most likely be closer to ng/mL than μg/mL, so go ahead and convert them.
- Finally, address the following in your report:
- which samples had a measurable amount of collagen I? collagen II?
- for samples with both values in range of the assay, what was the collagen II:I ratio?
- how do these results compare to those at the transcript level? what factors might cause any differences that you see?

### Part 2: Complete report

Your Module 3 report is due before you leave lab today. Please print a hardcopy of the report (double-sided, please!) in addition to emailing it in to the 20109.submit address.

### Part 3: Prepare for presentation

Next time you will present your research proposals to the rest of the class. Now is a great opportunity to get feedback from your peers and/or the teaching faculty. If you did not complete your cross-group research discussion last time, remember to do so today.

## For next time

Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question, and what you hope to learn. More detailed descriptions of the elements of the oral presentation can be found in the FNT assignments and the protocols associate with this Module as well as with the research proposal guidelines. When it is ready, please email your presentation to 20109 DOT submit AT gmail DOT com. **Speaking order will be determined by the order that presentations are received.**

On the day you present (see announcements on front page for when and where) your team should print out and bring **two copies** of your powerpoint slides. Black and white is fine and you can print more than one slide per page if you like (4-6 slides per page tends to be ideal for my note-taking). You should also write (and print out) your "talking points" in the comments box of each slide. These are speaking notes for your presentation, and should include short phrases to remind you of the key points to cover on each slide, as well as the transitions you've planned between them. For example, in a previous year's presentation one slide's talking points were:

*As you can see from this image, taken from a review on hydrocarbon metabolism in marine bacteria, the alcanivorax species is the first to grow in population after an oil spill, and its growth correlates with a decrease in aliphatic hydrcarbons.
*

- After most alkanes have been degraded, the Cycloclaticus species blooms while aromatic hydrocarbon levels decrease
- One thing to note is that as soon as they have done their job, both species return to their normal population levels.
- One problem with using Alcanivorax and Cycloclasticus to clean oil spills, however is that they can only be found in specific locations

The next slide (transition statement) began:
*To remedy this, we decided to look into other bacteria into which we could move the hydrocarbon metabolic pathways*

You don't have to use complete sentences in your own talking points, but the above example should give you a sense of what content is expected.

**You will be graded on the integrated success of your presentation: concepts, slides, talking points, and presentation.**

Next Day: Student presentations

Previous Day: Transcript-level analysis