Western Blot图片灰度分析 -Photoshop.pdfVIP

Western Blot 图片灰度分析 -Photoshop 2010 年02 月21 日 星期日 08:10 Comparing the intensity of bands on a Western blot can be done in a number of ways using software that is commonly found on lab computers or freely available for download. The following document outlines some of those methods. For a long time, the de-facto standard for analysis in labs that didnt want to spring for multi-thousand $$ commercial densitometry software was Adobe Photoshop or one of the competing photo editing programs. All you really need in a program is a freehand selection tool and a way to measure the mean gray value inside the selection. Well start with Adobe Photoshop as an example (you can find many references in the literature that include phrases such as densitometry was carried out in Photoshop in the methods section). To start with, youll need to scan in your xray film on a flat-bed scanner. This can be a cheap consumer unit, or a more expensive transparency scanner if you have access to such a beast. You can scan the film as a grayscale image, and set the resolution to a medium value (300-400dpi). 1. 1. 11.. Open the scanned image in Photoshop. 2. 2. 22.. Under ImageMode, check the grayscale option if its not already selected. We dont care about color information, only grayscale information, so we can discard the color information. 3. 3. 33.. Under ImageAdjustments, select Invert (or press Ctrl+I). Now the dark parts of the film are light, and the light parts are dark. This is useful later, as the high-expression bands, which are dark on the film, will have high numerical values when we measure them. When photo programs report darkness/lightness values, the dark points have values near zero, and the light points have values that max out at 255. Theinvertedscan 4. 4. 44.. Choose the lasso tool from the tool palette. 5. 5. 55.. On the first band, use the lasso tool to draw a line all the way around the edges of the first band. This is where your judgem