alpha Tubulin Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Tubulin is the major building block of microtubules. Microtubules function as structural and mobile elements in mitosis, intracellular transport, flagellar movement and in the cytoskeleton. The tubulin superfamily is composed of six distinct families. Genes from the alpha, beta and gamma tubulin families are found in all eukaryotes. Microtubules of the eukaryotic cytoskeleton perform essential and diverse functions and are composed of a heterodimer of alpha and beta tubulins. Alpha and beta tubulin dimers are assembled to 13 protofilaments that form a microtubule of 22 nm diameter with a theoretical molecular weight of 50 kDa.

Tyrosine ligase adds a C-terminal tyrosine to monomeric alpha tubulin. Assembled microtubules can again be detyrosinated by a cytoskeleton associated carboxypeptidase. Detyrosinated alpha tubulin is referred to as Glu-tubulin. Another post-translational modification of detyrosinated alpha tubulin is C-terminal polyglutamylation which is characteristic for microtubules in neuronal cells and the mitotic spindle.

Like GAPDH and beta-actin, alpha/beta tubulin is often used as a loading control in immunoblot applications (1). Alpha/beta tubulin is also good for counterstaining microtubules in immunofluorescence (2).

References

1. Hannen, R., Selmansberger, M., Hauswald, M., Pagenstecher, A., Nist, A., Stiewe, T., . . . Bartsch, J. W. (2019). Comparative Transcriptomic Analysis of Temozolomide Resistant Primary GBM Stem-Like Cells and Recurrent GBM Identifies Up-Regulation of the Carbonic Anhydrase CA2 Gene as Resistance Factor. Cancers (Basel), 11(7). doi:10.3390/cancers11070921

2. Nel, M., Joubert, A. M., Dohle, W., Potter, B. V., & Theron, A. E. (2018). Modes of cell death induced by tetrahydroisoquinoline-based analogs in MDA-MB-231 breast and A549 lung cancer cell lines. Drug Des Devel Ther, 12, 1881-1904. doi:10.2147/dddt.S152718

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-17427

• alpha Tubulin Antibodies
• alpha Tubulin Lysate
• alpha Tubulin Proteins