Protein Extraction and Western Blot

Total cell lysates were prepared for western blot by adding 2× Laemmli buffer directly to the wells in a 24-well plate followed by denaturation at 100°C for 5 min. SDS-PAGE and detection of proteins were carried out as described elsewhere (26 (link)). The following commercial primary antibodies were used (1:1000 dilution): FUS (rabbit polyclonal, 11570-1-AP, Proteintech); NONO (rabbit polyclonal C-terminal, Sigma); SFPQ (rabbit monoclonal, ab177149, Abcam); beta-actin (mouse monoclonal, A5441, Sigma).

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An H., Elvers K.T., Gillespie J.A., Jones K., Atack J.R., Grubisha O, & Shelkovnikova T.A. (2022). A toolkit for the identification of NEAT1_2/paraspeckle modulators. Nucleic Acids Research, 50(20), e119.

Publication 2022

ab177149 beta-actin

Antibodies Beta actin Cell Dilution Laemmli buffer Mouse Proteins Rabbit Sds page Western blot

Corresponding Organization : University of Sheffield

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of FUS, NONO, SFPQ, and beta-actin

control variables

Cell lysate preparation method (adding 2× Laemmli buffer directly to the wells in a 24-well plate followed by denaturation at 100°C for 5 min)
SDS-PAGE and protein detection procedures (as described elsewhere (26))

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

Annotations

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