Why fractionate?

Example:

Picture shows 10 µg of cellular fractions from Arabidopsis thaliana, separated on an 8-12% gradient gel. Target protein band was not detected in total leaf extract (1), but appeared as a strong band in photosysten I fraction (4).

Total leaf (1)
Chloroplast (2)
Thylakoid (3)
Photosystem I (4)

If no signal is detected in a total cell extract, it is worth fractionating, to obtain specific chloroplast, mitochondria or nuclei enriched fractions. This procedure will concentrate a target protein and lead to the detection of a desired band (for non-cytoplasmic proteins). This is shown in the example to the right - from no band in a total cell extract, to a specific band in the photosystem I fraction.
This approach is especially important when working with proteins of low expression. Fractionation, combined with using a specific organ, e.g. the root tip, or a leaf at a certain developmental stage, will greatly contribute to the successful identification of your protein of interest.

Examples of protocols to fractionate can be found here.
For Agrisera Western Blot Guide and video tutorials, check here.
In case of any questions, you are always welcome to contact Agrisera Technical Support.

Why fractionate?

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