To begin the quantification process, the first step is to upload the image into the image software. Once the image is uploaded, it may appear with some imperfections, such as bands that are not aligned or a background that needs to be removed.Įditing the Image: Inverting and Rotating There are two ways to do this - either by going to "File" and selecting "Open" or simply dragging the image into the software. To correct any alignment issues with the bands, we can use the editing functions of the image software. First, we can invert the image by going to the "Edit" menu and selecting "Invert." This will ensure that the bands appear in a straight line. If the bands are still slightly tilted, we can use the "Transform" option in the "Image" menu to rotate the image by a specific degree. Experimenting with different rotation angles will help achieve the desired alignment. In addition to aligning the bands, it is often necessary to remove the background from the image to obtain clear curves for analysis. This can be done by adjusting the brightness and contrast of the image. Using the "Adjust" option in the "Image" menu, we can navigate to "Brightness and Contrast" and make adjustments until the background is no longer visible. It may require some trial and error to find the optimal settings. Once the image has been edited to perfection, we can proceed to select the bands of interest for analysis. This is done by using the selection tool, often represented by a rectangle icon. ![]() The selection should encompass the bands while avoiding unnecessary areas. It is crucial to ensure that the selection includes all the bands of interest and that there is enough room underneath for further analysis. Once the selection is made, we can proceed to the analysis phase. The next step in quantifying Western blots is calculating the ratios. ![]() This involves dividing the intensity values of the bands by a control value. In this case, the control value is derived from the housekeeping or actin bands. By dividing the intensity values of the bands of interest by the control value, we can obtain ratios that represent the relative expression levels. Normalizing the Protein of Interest to Housekeeping Proteins Calculation of ratios should be performed for both the housekeeping genes and the protein of interest in both the control and treatment groups. pone.To normalize the protein of interest, we divide its ratio by the ratio of the housekeeping proteins. Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. WB_Sup_7days_for_Densitom.png -> Prepared image for densitometry analysis of supernatant samples WB_Lysa_7days_for_Densitom.png -> Prepared image for densitometry analysis of lysate samplesĢ0171219 WB_Sup_7days_for_Densitom.png -> Correlation plot of fluorescence and pixel intensity for supernatant samples ![]() JPG -> Raw western blot image for supernatant samplesĢ0171219 Fluor_Densito_Correlation_Lys.png -> Correlation plot of fluorescence and pixel intensity for lysate samples Supernatant samples were obtained by spinning 1 mL sample at 15000 × g for 10 min and transferring 100 μL from each well to the clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA), followed by fluorescence measurement.Ġ21115_20171219 Correlation_Fluore_vs_Densito_mCherry_50mL_7_days.xlsx -> data points for fluorescence measurements and pixel intensity obtained with FiJI (Fiji Is Just ImageJ).Ġ22615 WB SP mCHerry Lysate 3.JPG -> Raw western blot image for lysate samplesĠ22615 WB SP mCHerry Supernatant 3. Fluorescence was measured at excitation 575/9 nm and emission 608/20 nm. ![]() Then, 100 μL of each strain was transferred to a clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA) and fluorescence was measured using an Infinite® M200 PRO plate reader (Tecan, Männedorf, Switzerland). Image present in this dataset were obtained by following the steps described in the protocol dx.doi.org/10.17504/protocols.io.kfpctmn.įluorescence measurements were obtained by growing cc1690 in 50 mL TAP media on a rotary shaker, set to 150 rpm, under constant illumination (50 μmol photons/m 2s). The results present in this dataset were obtained from recombinant Chlamydomonas reinhardtii expressing the fluorescent protein mCherry in different constructs formats, aiming to recombinant protein secretion.
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