12/29/2023 0 Comments Logger pro absorbance![]() To calibrate the device, choose Calibrate > Spectrometer from the Experiment menu.To use the spectrometer in absorbance mode the spectrometer must be calibrated:.Wavelength will be displayed of the visible spectrum in nanometers superimposed over the colors associated with those wavelengths. The software will automatically identify the spectrometer.Device drivers were included with the Logger Pro 3 software. Note: Do not go online for device drivers.The first time you connect a SpectroVis Plus, your computer may ask you a few questions. Connect the spectrometer to the computer via a USB port.Requires Logger Pro 3 (Version 3.8.2 or higher) when using with a computer. Go to for the complete instructions or see the hard copy in the prep room. These instructions are taken from the Specifications sheet provided by Vernier. The OD ses of the undiluted protein is 1.673 The ODof a 1:10 dilution of your protein is 0.875 I First decide wliides of these two data points to use (1.673 or 0.Vernier Spectro-Vis Plus Spectrophotometer Overview The first, is an undiluted protein sample the second is a 1:20 dilution, made by adding 100u of original Nettie protein to 9001 of visulled water. 0.4 Regression Equation: y = 0.9429x + 0.1196 8 : 0 0.5 2 Concentration (mg/ml) You make two versions of the Nettie protein to measure via the Bradford Assuy. In a Bradford Assay, you generate the following standard curve and regression equation: Ooms 12 2. You are trying to determine the concentration of the protein Nettle that you have isolated by ammonium surate predpitation followed by size exclusion chromatography. Absorbance 1.4 1.2+ Maximum Absorbance Graph plateaus due to all the dye being bound by protein Absorbance A (00) 0.8 0.61 Maximum protein concentration 0.4 0.2 10 15 Concentration / Absorbance 1.4 12 Maximum Absorbance Graph plateaus due to all the dye being bound by protein sorbance A 100 0.8 MaximurÄ”.0 Bradford Assay Standard Curve Protein concentration v. This concept is illustrated by the graph below: 16 Bradford Assay Standard Curve Protein concentration v. That is, you can increase the protein level to a point where available dye becomes exhausted so the graph would plateau. As the dye is limiting (i.e., you only put in a set amount), then you can reach a point when all the dye is bound, and no Š° matter how much more protein you put in you will not see a change in absorbance. That is, the Bradford assay is really measuring the amount of bound dye and NOT the amount of protein present (although the two are connected until the dye is all used). The Bradford assay is based on a shift in the absorbance of the dye, Coomassie Brilliant Blue G-250, from 465 nm to 595 nm when it binds a protein under acidic conditions. The assay is an indirect (or colorimetric) method for estimating protein concentrations. The Bradford assay has limits, it does not work for all protein concentrations. In which term (% transmission or absorbance) are the data most usefully expressed? Why do we tend to use one set of values and not the other? (Hint: it's a math thing) 1 ![]() What is the difference between % transmission and absorbance? 6. Spectrophotometers can measure either %transmission or absorbance. howhich wavelength would be the best one to use to measure the concentration of protein in a solution? Why this wavelength and not another wavelength 100nm higher or lower? 5. Below is the absorption spectra for Coomassie Blue - the dye involved in the Bradford Assay. What are the concentrations of the 2 unknown hemoglobin samples that you measured in lab? Use (and show your work) the regression line equation to calculate the concentration of the unknown solution. What do the different values in the equation represent? 3. Record the equation for the regression line. Description - see: Writing Papers in the Biological Sciences for good examples of legend writing. Paste in a labeled copy of your graph as a figure here in your answer and write an appropriate descriptive legend below it (Figure 1). Then insert a trend line (linear regression line) to form the standard curve. ![]() Use Logger Pro to generate a standard curve for the BSA standards that you measured in the spectrophotometer.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |