Friday 26 November 2010

GGS LIVE - Immunofluorescence

Yo Biofreakers:)

Today in the GGS LIVE section we are going to look at the immunofluorescence staining technique.

Method: Immunofluorescence.

About: Immunofluorecence is a technique that allows for detection of a particular protein within the cell.

What: Staining for gamma-H2AX and Rad51 proteins in chicken DT40 cells after ionizing radiation treatment.

Immunofluorescence technique is perfect for studying protein localization. Staining procedure is not compilcated and a representative protocol is shown on the cartoon below:

Briefly, DT40 suspension cells are harvested by centrifugation. Media is discarded and cell pellet is resuspended in 1 x PBS (phosphate buffered saline). Cells are adhered to poly-L-lysine slide, fixed and permeablised to allow access of molecules used in subsequent steps. Next, slide is incubated in blocking solution (usually a neutral protein, like 1 % bovine serum albumin solution). Blocking protein binds to sticky spots on slide, cells and within cells in order to decrease unspecific binding of the primiray and secondary antibodies (reduce unwanted background signal). After blocking, slides are incubated with primary antibodies that recognises the protein of interest. Notice that primary antibody has higher affinity to protein of interest and displace blocking protein. Slides are then washed to remove unspecifically bound antibodies. Later, secondary antibodies conjugated to a fluorophore are added and again washes are performed to remove unspecifically bound antibodies. Complex protein of interest - primary antibody - secondary antibody is formed and protein is ready to be detected and analysed by microscopy.
Lets have a look at our results.

As you can see in our study case experiment, cells treated with ionizing radiation were stained against two different proteins. Gamma-H2AX is a phosphorylated form of H2AX histone. H2AX histone is phosphorylated in proximity of DNA double strand break, appears as fast as 1-2 min after radiation and persists few hours post-treatment. Rad51 is a DNA repair protein and it accumulates at site of damage 2-4 hours after IR treatment. As you can see from our results both gamm-H2AX and Rad51 proteins form foci within nucleus (representative foci are indicated with yellow arrowheads). Both proteins nicely colocalise as seen on the overlay picture. With such immunofluorescence staining we can follow foci formation, resolution, change in protein localizatio or etc.

I hope you enjoyed:)



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