Q: How do I get the protein concentration in mg/ml from the standard curve if the X-axis is in units of µg.

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1 Photometry Frequently Asked Questions Q: How do I get the protein concentration in mg/ml from the standard curve if the X-axis is in units of µg. Protein standard curves are traditionally presented as Abs vs. protein mass, µg. The equation of the line for the standard curve allows you to use the absorbance value read from an assay of the unknown concentration as Y, to solve for X, which has units of mass, µg. The parameter to be determined, however, is the concentration of the unknown solution. Concentration is mass per unit volume. So, the volume used to compute the concentration must be either A) the volume of the diluted protein sample used in the assay (500 or 120 µl), then that answer multiplied by the factor by which the unknown was diluted prior to transfer to the assay tube; OR B) the equivalent volume of undiluted unknown solution transferred to the assay tube, which is one-fourth of the volume of unknown that was added to water. For example, imagine the triplicates of tube 8 of the Lowry assay had an average absorbance that calculated to 100 µg protein. A) The unknown solution of protein in tube 8 was diluted by (2000 µl final volume / 60 µl unknown). The unknown protein concentration then is determined to be (100 µg / 500 µl) x (33.33) = 6.67 µg/µl) B) Alternatively, the diluted protein solution was a total of 2.0 µl, and only one-fourth of that, or 0.5 µl, was used in each assay tube. So, the equivalent volume of undiluted solution put into each assay is one-fourth the amount of protein solution (60 µl) put into tube 8, or a total of 15 µl. Then, the concentration of the unknown solution is determined to be 100 µg / 15 µl = 6.67 µg/µl. Q: Where we need to determine the extinction coefficient, I was wondering what we should use for concentration units. Normally, we use M or mm, but I was wondering if mg/ml is fine also as long as Absorbance is unitless and everything is consistent. If not, where should I find the molecular weight of BSA? The extinction coefficient must have the inverse of the unit of concentration that was given for the standard solution used to measure the absorbance from which the extinction coefficient is calculated. The units are either (µg/µl) -1 cm -1. The molecular weight of BSA is commonly given in tables, calculated from the amino acid sequence, and used as SDS-PAGE MW markers as the molecular mass of denatured protein, 67 kda. This is the subunit MW. You had native BSA (non-denatured, or intact) in the cuvette. Do you know whether BSA is a monomer or a homo-multimer? Interaction between subunits in solution may quench the absorbance of light by the side chains that are responsive. Side chain interactions may also quench absorbance within a native monomer relative to the completely denatured, structure-less amino acid chain. Extinction coefficients calculated by computer programs from the molar percentage of absorbing side chains and their corresponding molar extinction coefficients are also assuming the protein is denatured in solution. The crystal structure of human serum albumin has been solved. A cartoon of the structure is shown below with separate chains in different colors. 1

2 Q: How can I use KaleidaGraph to analyze my data? First arrange your standard protein mass and triplicate absorbance values in columns and label them appropriately. Use "Formula Entry" to calculate the mean value of the three trials. You can choose "Windows" and select "Formula Entry" from the pull-down menu, or you can simply click on the formula entry box if it is visible on your screen. Choose any one of the tabs (F1 thru F8) to enter your formula. Type "c4=mean(1:3)" and click "Run". This will calculate the mean of the values in columns 1 through 3 and input this value into column 4. Remember that KaleidaGraph lists the first column as "c0." Now you need to calculate the standard deviation of your data in order to add the error bars. Choose another formula tab and type "c5=std(1:3)" and click "Run." This will calculate the standard deviation of the three columns of data and input the value into column 5. Q: How can I use KaleidaGraph to prepare my graph? Now you can construct a scatter plot, following the same steps as outlined in Example 2 ("Creating a Scatter Plot with Linear Curve Fit and Error Bars") of the KaleidaGraph tutorial. You need to follow a different series of steps, though, to create the error bars. After preparing your scatter plot and performing the linear curve fit, select "Error Bars" under the "Plot" menu. On the pop-up window, click on the checkbox for the Y variable -- this will open a new window. Under "Error type" choose "Data Column" and select the column that contains the values for standard deviation (probably column 5) and click "OK." Now click "Plot" to get your error bars. If you want to modify the text for your axis labels, you can double click on the label and the format window will open. If you want to change "ug" in your X-axis label to include the Greek symbol (µg), you can highlight the letter "u." Then choose "symbol" under the "Font" menu and type in the letter "m" for the Greek symbol mu. If you want to modify the Y-axis label to produce a subscript of the wavelength number, highlight the number (750 or 595) and select "subscript" from the "Style" menu. If you want to rotate the Y- axis label, you can select "0 o Rotation" under the "Format" menu. 2

3 Here is a sample of what your graph should look like: Q: How can I use KaleidaGraph to print my graph and data? You can simply select "Print Graphics" under the "File" menu, or you can prepare a layout that includes more than one graph and print that. The steps for preparing a layout are specified in Example 4 (page 10) of the tutorial. To print your data, simply select "Print Data" under the "File" menu. 3

4 Q: What are the values and units for the standard curve? First, look at the following table, which lists both the concentrations of diluted protein standards 1 through 6 for the Lowry assay as well as the masses of protein assayed per trial. tube Volume H 2O, ml Volume standard (1 mg BSA/ml), l total volume diluted standard solution, ml g BSA in diluted solution concentration diluted standard, g/ml possible independent variable Volume diluted protein assayed, ml Mass protein assayed, g Assay data A A preferred independent variable A mean A dependent variable To graph these data, the X values may be either the masses of protein assayed or the concentrations of diluted standard. The Y values must be the mean absorbances. It might be easier or make most sense to use the concentrations of diluted standard as X values, though some people get confused because the standard has a known concentration of 1 mg/ml. Thus, it would be necessary to plot the concentrations of the several diluted solutions, which in this case all have a total volume of 2.0 ml. Because Lowry originally published his standard curve as absorbance versus mass, protein standard curves are most commonly presented as absorbance versus mass assayed. In each of the triplicate assay tubes, only 0.5 ml of diluted protein solution was used, so only one-fourth of the protein put into each of tubes 1-10 was actually assayed. 4

5 Notice that the two graphs above look very similar. The important difference is in the values and units of the slopes in the equations of the lines used to calculate the X values for the unknown from the recorded absorbance values (Y values). For the graph on the left, the answer will be X = protein mass in mg, while on the right, the answer will be X = protein concentration in mg/ml. In order to obtain a concentration of unknown from the left-hand graph, for each trial you must divide the calculated mass by the volume of unknown protein solution that was assayed in each tube. Q: How do I include error bars in an Excel plot? Include a data column in your spreadsheet that calculates the standard deviation of duplicate absorbance values. Plot your average or mean values of absorbance as a scatter plot and add the required trend line and R2 value. Select the data series by clicking on any one of the data points. Right click on the series and select "Format data series." On the pop-up window, select the tab marked "Y Error Bars" and select the option marked "Custom." Use the grid icon to the right of the text box to select the data column containing the standard deviation values. You will need to select this column for both the (+) and (-) error bars. Make sure you have selected "Both" under the "Display" heading. Click OK to plot the error bars. 5