WesternBlot图片灰度分析-Photoshop

Western Blot图片灰度分析–Photoshop

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 didn't 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. We'll 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, you'll 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.Open the scanned image in Photoshop.

2.Under Image>Mode, check the grayscale option if it's not already selected. We don't care

about color information, only grayscale information, so we can discard the color information.

3.Under Image>Adjustments, 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.

The inverted scan

4.Choose the lasso tool from the tool palette.

5.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 judgement comes in to play, determining where the edges of the band are, and what is simply background.

6.For Photoshop CS2/3: If the histogram window is not open by default, go to

Window>Histogram to open the histogram. In newer versions of Photoshop (CS2, CS3) there is a small arrow in a circle in the upper right corner of the histogram window. Click on this and choose 'expanded view' to show the values for your selection.

For Photoshop v.5+6: Go to Image>Histogram to display the histogram for the current selection.

6.The histogram information includes a "Mean" value and a "Pixels" value. Record these

two numbers for your selection. The Mean value is the average gray value (from 0 to 255) for the area inside your selection. The Pixels value is the number of pixels contained in your selection area. Bands with high expression are typically darker, but also often larger in size, so we want to know both of these attributes for our comparison later.