Immunoblot Analysis of Cas9 and Ubiquitin

Cells were lysed in RIPA buffer and immunoblot analysis was performed as previously described [45 (link)]. Briefly, total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with the appropriate antibodies. Anti-Cas9 (1:500; #517386; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Ub(P4D1) (1:500, #8017, Santa Cruz Biotechnology), or anti-α-Tubulin antibodies (1:500, #32293, Santa Cruz Biotechnology) were used as primary antibodies and HRP-conjugated goat anti-mouse IgG (1:10,000; #ADI-SAB-100-J; Enzo Life Sciences, Farmingdale, NY, USA) was used as the secondary antibody.

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Han S.W., Jung B.K., Park S.H, & Ryu K.Y. (2019). Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System. International Journal of Molecular Sciences, 20(13), 3168.

Publication 2019

Anti-Cas9 anti-Ub(P4D1) anti-α-Tubulin HRP-conjugated goat anti-mouse IgG #ADI-SAB-100-J

Anti antibodies Anti igg Antibodies Antibody Buffer Cell Goat Immunoblot Mouse Ripa Sds page α tubulin

Corresponding Organization : University of Seoul

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Variable analysis

independent variables

Cas9 expression
Ubiquitination (Ub)

dependent variables

Protein expression levels of Cas9 and Ub

control variables

α-Tubulin expression (used as a loading control)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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