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How do you quantify a Western blot?

How do you quantify a Western blot?

Step 1: Determine the background-subtracted densities of your protein of interest (PI) and the normalizing control (NC). Step 2: Identify the NC that has the highest density value. Step 3: Divide all the NC values by the highest NC density value to get a relative NC value.

How do you quantify blots in ImageJ?

You can quantify by the following steps:

  1. Open western image in Image J, select Rectangular Selections tool from the ImageJ toolbar and select first western band.
  2. Press Ctrl +1 and drag the same rectangle selection to the next band and press Ctrl + 2.

How do you analyze a Western blot in ImageJ?

1. Open the western blot image in ImageJ. 2. Make sure that the image is in 8-bit mode: go to Image>Type>8-bit….Data analysis

  1. Place your data in a spreadsheet.
  2. In a new column next to the Percent column, divide the Percent value for each sample by the Percent value for the standard (the 1st peak in this case, 26.666).

How do you quantify in ImageJ?

From the Analyze menu select “set measurements”. Make sure you have area integrated intensity and mean grey value selected (the rest can be ignored). Now select “Measure” from the analyze menu.

Why is washing step essential for Western blot?

Washing, blocking and antibody incubation Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically. It is often a good strategy to incubate the primary antibody with BSA since it is usually needed in higher amounts than the secondary antibody.

How do you do a densitometry in Western blot?

Densitometry Western Blot

  1. Open Western scan in Image.
  2. Click the ‘rectangular selection’ (under file menu) and select a box around a band of interest.
  3. Once you have boxed an area go to the ‘analyze’ drop menu.
  4. Use the arrow curser to move your box along to the next band of interest, and repeat the ‘measure’.

How do you analyze dot blots?

There are two built in methods for analyzing a dot blot in ImageJ. The first is to treat each row as a horizontal “lane” and use ImageJ’s gel analysis function. The second is to subtract the background and measure the integrated density of each dot.

How do you do a densitometry in western blot?

How do you normalize western blot data?

To perform Western blot normalization using a single protein as a control, the blot is probed with a primary antibody specific for the protein of interest, and one directed against a normalization control. Ideally, the normalization control is a protein that is present at constant levels in every sample.

How do you quantify fluorescence intensity?

Determining Fluorescence Intensity and Signal

  1. To threshold your image, go to Image > Adjust > Color threshold. Slide the Hue slider to match the color- so that the fluorescent areas are selected.
  2. Go to Analyze > Analyze Particles > Display results.
  3. Add areas for all fluorescent regions.

How does ImageJ calculate area?

To measure the area of water that you highlighted in each of the three images, select the Rectangular Selections tool in the ImageJ toolbar and drag a rectangle over just the top image of the triptych. Choose Analyze > Set Measurements… and click the Area and Limit to Threshold checkboxes.

What are the steps in western blotting?

Five steps are involved in western blotting procedure and detection assay, namely, transfer, blocking, primary antibody incubation, secondary antibody incubation and protein detection, and western blotting analysis.