Staining Actin and Tubulin – from WB to Live Cell Imaging

Anti Anti Ab - IF photo

Fig. 1: Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody (027AAN01).

Actin and Tubulin, as the major cytoskeleton structures, are crucial components of a plethora of processes in cell biology. Both are very much involved in stabilizing the cell shape, and especially in cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Thus visualizing Actin and Tubulin in fixed or living cells and detecting them in biological samples (e.g. in Western Blots) belongs to basic experimental set-ups in Cell Biology.

A broad range of tools is available for all kind of experimental levels, in this post we’ll take a look at some of these you can use from Western Blot to Live Cell Imaging.

Detection of Actin and Tubulin with Antibodies

Anti actin Ab - WB Photo

Fig. 2: Western blot of purified actin and cell extracts probed with anti-actin antibody (027AAN01).

 

Antibodies can be used in diverse methods, such as Western Blots, ELISA, and Immunocytochemistry. Good results in different methods can be only obtained with a few antibodies.

Anti pan Actin has been thoroughly tested in these application and brought brilliant results (See Fig 1 and 2).

Anti tubulin Ab - IF photo

Fig. 3: Immunofluorescence image of Arabidopsis thaliana metaphase cell stained with Anti-Tubulin polyclonal antibody (027ATN02).

Anti Tubulin Ab WB photo

Fig. 4: Western blot of cell extracts probed with anti-tubulin polyclonal antibody (027ATN02)

 

In addition, here you can browse a selection of other anti Actin antibodies .

Anti-alpha/beta tubulin has been positively tested in the same applications and can additionally be used for Immunoprecipitation experiments. Results are presented in Fig. 3 and Fig. 4.

Here’s a complete overview of a range of anti Tubulin antibodies.

Actin staining of fixed cells

Actistain photo

Fig. 5: Swiss 3T3 fibroblasts stained with ActiStain 488 (green), Dapi (blue) and Anti Vinuclin (orange).

In fixed cells Actin can be stained with antibodies (see above) but often fluorescent phalloidins are used. Phalloidin belongs to the group of phallotoxins produced by the mushroom Amanita phalloides (death cap mushroom). The natural toxicity of Phalloidin is due to its stabilizing effect on F actin in cells. Based on its affinity for F actin and coupled to a fluorescent dye, it can be used to visualize F actin.

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability. Results of staining of Swiss 3T3 cells with ActiStain 488 are shown in Fig. 5.

Live Cell imaging of Actin and Tubulin

SiR Tubulin Spherochrome tebu-bio's fluorescent dye

Fig. 6: 3D-SIM microscopy image of microtubules in primary rat cortex neuron body labeled with SiR-tubulin.

Finally, I would like to introduce brand new tools to stain Actin and Tubulin in living cells without the need to transfect them with vectors carrying the information for fluorescently labeled Actin/Tubulin or binding proteins. Spirochrome recently released unique fluorescent stains for monitoring both major components of thecytoskeleton easily in living cells. These fluorescent stains (SiR-Actin and SiR-Tubulin) are cell permeable compounds which stain F-actin (and microtubules, respectively; see Fig. 6 and Fig.7).

3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts.

Fig. 7: 3D-SIM microscopy image of Actin stress fibers in human primary dermal fibroblasts labeled with SiR-actin.

 

To learn more about these exciting stains,  take a look at my recent post 2 new Actin and Tubulin live-cell imaging stains – without transfection!

 

Interested in staining Actin and/or Tubulin? Get in touch by leaving your questions or comments below!

 

 

 

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