Creating Optimal Conditions for OPA1 isoforms by Western Blot in Muscle Tissue

Stephens, D. C.; Crabtree, A.; Beasley, H. K.; Garza-Lopez, E.; Mungai, M.; Neikirk, K.; Vang, L.; Vue, Z.; Vue, N.; Marshall, A. G.; Turner, K.; Shao, J.-q.; Murray, S. A.; Gaddy, J. A.; Wanjalla, C.; Davis, J.; Damo, S. M.; Hinton, A.

doi:10.1101/2023.05.20.541601

bioRxiv · 10.1101/2023.05.20.541601

Creating Optimal Conditions for OPA1 isoforms by Western Blot in Muscle Tissue

Stephens, D. C.·Crabtree, A.·Beasley, H. K.·Garza-Lopez, E.·Mungai, M.·Neikirk, K.·Vang, L.·Vue, Z.·Vue, N.·Marshall, A. G.·Turner, K.·Shao, J.-q.·Murray, S. A.·Gaddy, J. A.·Wanjalla, C.·Davis, J.·Damo, S. M.·Hinton, A.

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Abstract

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1s ability to control mitochondrial functions. However, isolating OPA1 all long and short isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate 5 different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function. Tweetable AbstractWestern blot protocol optimization to visualize OPA1 isoforms. HighlightsO_LIProtocol for isolating OPA1 isoforms in primary skeletal muscle myoblast and myotubes C_LIO_LISteps for running isolated skeletal muscle cells from muscle tissue on a gel C_LIO_LIHow to collect samples in preparation for western blotting C_LIO_LIDetection of OPA1 isoforms C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/541601v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1dc254dorg.highwire.dtl.DTLVardef@150d3eorg.highwire.dtl.DTLVardef@186ed2org.highwire.dtl.DTLVardef@13cc3a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1c518ceorg.highwire.dtl.DTLVardef@df826borg.highwire.dtl.DTLVardef@1eb5bf8org.highwire.dtl.DTLVardef@e324e6org.highwire.dtl.DTLVardef@8baedc_HPS_FORMAT_FIGEXP M_TBL C_TBL Method SummarySamples for western blot analysis are isolated from lysed cells, loaded onto a gel, and ran using optimized conditions to better isolate OPA1 isoforms. Samples are transferred to a membrane for incubation and protein detection using OPA1 antibodies.

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Stephens, D. C., Crabtree, A., Beasley, H. K., Garza-Lopez, E., Mungai, M., Neikirk, K., Vang, L., Vue, Z., Vue, N., Marshall, A. G., Turner, K., Shao, J.-q., Murray, S. A., Gaddy, J. A., Wanjalla, C., Davis, J., Damo, S. M., Hinton, A.. 2023-05-22. Creating Optimal Conditions for OPA1 isoforms by Western Blot in Muscle Tissue. https://doi.org/10.1101/2023.05.20.541601

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