20.109(S08):Wrap-up analysis (Day7): Difference between revisions
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#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. | #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. | #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 | #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.) | ||
#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). | #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). | ||
#Click in the chart area, then select Chart | #Click in the chart area, then select ''Chart'' → ''Add Trendline''. Click on the Options Tab, and choose to display both the equation and the R2 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 R2 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 R2 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. The $ symbol in Excel is useful here for efficient calculations. | #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. The $ symbol in Excel is useful here for efficient calculations. | ||
Revision as of 08:54, 31 January 2008
Introduction
Protocols
Part 1: ELISA analysis
The analysis of protein concentration that you perform today will be similar to the titration curve analysis that you did in Module 2.
- 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.)
- 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).
- Click in the chart area, then select Chart → Add Trendline. Click on the Options Tab, and choose to display both the equation and the R2 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 R2 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. The $ symbol in Excel is useful here for efficient calculations.
- 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 notebook:
- 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: Prepare for presentation
Next time you will present your research proposals to the rest of the class. Try drafting at least 2 or 3 slides if you can. Atissa will come by to critique your slides and answer questions ~ 3(4?) pm.
For next time
-oral: finish
