Protein Expression Analysis of FKRP in Larval Tissues

Larval heads were removed and used for genotyping, bodies were snap-frozen, homogenised in immunoprecipitation buffer (Pierce). In all, 30 μg of protein was loaded on a 4–12% Bis-Tris Protein Gel MES Buffer (ThermoFisher Scientific). Primary antibodies were prepared in TBS blocking buffer (LI-COR): fkrp: rabbit polyclonal 1:500 fkrp^{48 (link)}, 1:500, Beta-Tubulin-Loading Control (Abcam: ab6046), Golgi 50 kda, IgG rabbit (Abcam: ab6046) 1:10,000, incubated in secondary antibody IRDye 800CW goat anti-rabbit (LI-COR) 1:5000. Blots were scanned on a infrared scanner (Odyssey). Co-immunoprecipitation utilised 10ug of antibody, fibronectin or FKRP, and a protein-G dynabead kit (Thermofisher).

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Wood A.J., Lin C.H., Li M., Nishtala K., Alaei S., Rossello F., Sonntag C., Hersey L., Miles L.B., Krisp C., Dudczig S., Fulcher A.J., Gibertini S., Conroy P.J., Siegel A., Mora M., Jusuf P., Packer N.H, & Currie P.D. (2021). FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy. Nature Communications, 12, 2951.

Publication 2021

TBS blocking buffer IgG rabbit IRDye 800CW goat anti-rabbit

A protein Antibodies Antibody Beta tubulin Bis tris Bodies Buffer Co immunoprecipitation Fibronectin Frozen Goat Golgi Heads Immunoprecipitation Irdye 800cw Larval Protein Protein g Rabbit

Corresponding Organization :

Other organizations : Australian Regenerative Medicine Institute, Monash University, Macquarie University, EMBL Australia, Fondazione IRCCS Istituto Neurologico Carlo Besta

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Variable analysis

independent variables

Removal of larval heads

dependent variables

Protein expression levels of fkrp, Beta-Tubulin, and Golgi 50 kda
Protein-protein interactions between fibronectin or FKRP

control variables

Protein loading amount (30 μg)
Gel type (4–12% Bis-Tris Protein Gel MES Buffer)
Primary antibodies (fkrp: rabbit polyclonal 1:500, Beta-Tubulin 1:500, Golgi 50 kda 1:10,000, IgG rabbit 1:10,000)
Secondary antibody (IRDye 800CW goat anti-rabbit 1:5000)
Immunoprecipitation buffer (Pierce)
Protein-G dynabead kit (Thermofisher)

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