imagej western blot quantification protocol

#IMAGEJ WESTERN BLOT QUANTIFICATION PROTOCOL HOW TO#

Therefore, three curves have been established.ġ2. In this example, we know that the protein consists of 3 subunits, and they represent the majority of the protein sample. Draw a line at the bottom of the peak that represents the first standard to define the area of the curve - Keep drawing all the single lines to define the curves in your standard lanes, and draw multiple lines in your lanes with your protein of interest. On the ImageJ interface, select the 'line' button The peaks (or valleys if your image is inverted like the one in this example) in each grid correspond to the intensity of the bands in the lane The standards lanes should only show one peak, while the lanes with protein you want to quantify will probably show multiple bands, as in the image below - Now is a good time to save! If the plot window is active, when you go to File→Save through the ImageJ interface, it will be trying to save your histograms if the image window is active, the image will saved. Once all lanes are defined, go to Analyze→Gels→Plot lanes (or use 'Ctrl+3') to generate histograms of each lane

#IMAGEJ WESTERN BLOT QUANTIFICATION PROTOCOL HOW TO#

If you are some kind of wizard who knows how to fix these mistake boxes without having to start over again, you should let me know!ġ0. You will get really good at marking the lanes, I promise. My best advice if you find yourself in that predicament is to close the file, reopen it and start again.

I think at this stage it's easiest to use key command 'Ctrl+2' to continue numbering the subsequent lanes (less of a chance to mess up!) - I do not know how to correct the inevitable mistake boxes that you are going to make by accident and that cannot be undone.I'm sorry! Deleting them will cause useless white space where the rectangle was previously. Repeat steps 7-8 until all lanes have been selected and numbered

Go to Analyze→Gels→Select next lane - Can also use key command 'Ctrl+2' - A tiny “2” will appear in the laneĩ. DO NOT DRAW A NEW RECTANGLE! You must drag the same rectangle you just made - The point here is to compare the band in each subsequent lane using the exact same size/white space/noise as the originally defined area in Lane 1Ĩ. Make sure your cursor shows as an arrow, grab the rectangle you just made, and drag it to the next lane Go to Analyze→Gels→Select first lane - Can also use key command 'Ctrl+1' - A tiny “1” will appear in the laneħ. Select the rectangle tool, and draw a box around the lane, making sure to include some of the empty gel between lanes and white space outside of the bandĦ. Find the lane with the lowest concentration of BSAĥ. Image→Adjust→Brightness/contrast - I recommend saving the image with an updated name at this point so that you have it to go back toĤ. Does your image look too dark or too light?.

Make sure you save your gel images as the same type of image (either. jpg (in case the tif file can’t be opened-an issue I am experiencing at the other lab).

After running and destaining the gel, take a picture and save it as a.

For 5GB1, BSA works great as a protein standard, and a range of 0.025 μg/μL to 5.0 μg/μL works well as a range for the standard curve

To determine protein concentration you will need to have a standard curve to compare your samples to.

Determining the concentration of protein in SDS-PAGE gel bands using ImageJ

Imagej western blot quantification protocol