Tuesday, 23 November 2010

GGS LIVE - Cell fractionation

Method: Cellular fractionation.

About: Cellular fractionation is a technique that allows analysing protein localization to different cell compartments.

What: Comparison of protein X and Y localization in wild-type and mutant cell lines.

There are many protocols for cellular fractionation. Selection of a particular fractionation protocol may depend on cell type used, required fractionation resolution or protein analysed. In our study case we will look at a very basic fractionation protocol for mammallian cells. Cartoon below represents majors steps of the protocol:

Briefly, cells are incubated in hypothonic buffer to facilitate cell lysis. Cell membrane of swollen cells is mechanically disrupted by action of dounce homogeniser. At this stage cellular organelles are spun and supernatant containing cytoplasmic proteins is saved. Nuclei are incubated (usually with rocking or rotating) in high salt and detergent containing buffer to extract nuclear proteins. Again cell remaining are spun and supernatant containing nuclear proteins is saved. In the last step DNA bound proteins are extracted either by sonication (DNA sharing) or DNAase treatment (DNA digestion). After solubilization of DNA bound proteins extracts are spun and supernatant containing DNA bound proteins saved. All obtained samples can be now analysed by a method of choice. In our study case we will analyse our samples by Western blotting. Equal volume of each sample is separeted by SDS-PAGE to retaing similar loading and proteins are detected by Western blotting (for Western Blot tutorial click GGS LIVE Western Blotting). Ok, lets have a look at our results. In our experiment we have fractionated wild-type and mutant cell lines. To be sure that protocol worked perfectly, we have to first look at proteins that are known constituents of each cellular fraction. Cytoplasmic protein beta-actin, DNA binding protein Histone H3 and nuclear protein nucleophosmin are only present in cytoplasm, chromatin and nuclear fractions, respectively. Additionally all control proteins are present in Whole Cell Lysate sample. This is an additionall control which tells us if our protein of interest was present in the starting material.

As you can see analysis of protein X and Y reveals their distinct localization between wild-type and mutant cell line. When Protein X is exclusively cytoplasmic within wild-type cell line, it is also present in the nucleus of the mutant cell line. On the other hand protein Y is nuclear and partially bound to DNA in wild-type but its exclusively cytoplasmic in mutant cell line.

I hope you enjoyed it.



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