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. jpg (in case the tif file can’t be opened-an issue I am experiencing at the other lab). Does your image look too dark or too light?.Make sure you save your gel images as the same type of image (either. Enlarge the image using the Magnifying Glass tool so you mainly just see the bands you want to measure. You have to do this again, because the powerpoint file saves it as an RGB tiff file. Convert the image to 8-bit (Image > Type > 8-bit). 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. HOW TO QUANTIFY WESTERN BLOT BANDS using ImageJ Area Under The Peak Method Adwoa Biotech. Open the TIFF file you created above in ImageJ. Find the lane with the lowest concentration of BSAĥ. 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. Western blotting has been a staple in life science labs for several decadesever since researchers published the first detailed description of this protein detection technique in 1979. 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. 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. The signal intensity of the band is directly. 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. Quantitating a western blot refers to the measurement of the signal emitted by your protein band(s) of interest. Errors can arise from many sources including sample preparation, unequal sample loading, or uneven transfer, such as when bubbles prevent protein transfer from the gel to the membrane 1 2 3. 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. To effectively determine the relative amount of a target protein on a blot, a normalization control is needed. My best advice if you find yourself in that predicament is to close the file, reopen it and start again. You will get really good at marking the lanes, I promise. Once all lanes are defined, go to Analyze→Gels→Plot lanes (or use "Ctrl+3") to generate histograms of each lane 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.
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