Reusch S et al. (2020), Affinity Purification of Label-free Tubulins fr...

XB-ART-57667

STAR Protoc 2020 Dec 18;13:100151. doi: 10.1016/j.xpro.2020.100151.

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Affinity Purification of Label-free Tubulins from Xenopus Egg Extracts.

Reusch S , Biswas A , Hirst WG , Reber S .

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Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications. The protocol is applicable to any cell or tissue of interest. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).

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Genes referenced: ckap5 mrc1 nhs

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	FigureÂ 1. Purification and Coupling Efficiency of GST-TOG1/2Expressed and purified GST-TOG1/2 (Input) used to make two TOG columns, flowthrough (FT) after conjugation to a NHS-column one (#1) and two (#2).
	FigureÂ 2. Overall Schematic WorkflowFor each stage (dotted box), steps are annotated.
	FigureÂ 3. Preparing Xenopus Egg Extract(A) Good-quality Xenopus laevis eggs.(B) Lysed and puffy eggs (in red squares).(C) Sorted eggs that have been washed with MMR Buffer.(D) After successful dejellying, eggs are densely packed.(E) Eggs transferred into a suitable centrifuge tube.(F) After a short low-speed spin eggs are tightly packed.(G) A subsequent high-speed spin is used to crush the eggs and fractionate them into different layers: layer I is enriched in lipids, layer II is critical as it contains all cytoplasmic proteins including tubulin and layer III contains a mixture of pigment granules, yolk, nuclei, and egg cortex.(H) With the help of a syringe and 18G needle, layer II is extracted.
	FigureÂ 4. Coomassie Staining and Western Blot Analysis of the Tubulin Purification(A) Coomassie-stained SDS-PAGE of samples taken during the purification of tubulin. (1) Lysate; (2) HS spin; (3) Flow-through; (4) 1st wash; (5) 2nd wash; (6) ATP wash; (7) 4th wash; (8) 5th wash; (9) 6th wash; (10) Pooled elutions; (11) Desalted and concentrated tubulin.(B) Western blot with antibody against Î±-tubulin of the same samples as in (A).
	FigureÂ 5. Posttranslational Modifications and Activity of Purified Tubulin(A) Western blot against tubulin PTMs before and after the purification. 300Â ng tubulin for each species (Bos taurus, Xenopus laevis, Xenopus tropicalis) were loaded onto a polyacrylamide gel adjacent to a corresponding volume of egg extract. Samples were transferred to a nitrocellulose membrane and probed with antibodies against Î±-tubulin, tyrosinated tubulin (Tyr), detyrosinated tubulin (Detyr), acetylated lysine 40 (K40), and phosphoserine (Pser).(B) Tubulin polymerization assay for assessing the relative activities of bovine brain tubulin and Xenopus tubulin purified via the TOG column. 2Â Î¼L of the 20Â Î¼M initial reaction mixture was loaded onto the gel as the input (I), followed by equivalent volumes for the supernatant (S) and the pellet (P) after resuspending the pellet in the same starting volume of 1Ã— BRB80.

References [+] :

Al-Bassam, Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP. 2011, Pubmed, Xenbase