Quantitative Immunoblotting Analysis of Asi2 Protein

Cycloheximide chase and immunoblot analyses were performed as described in Ref. 16 (link). Immunoblotting was performed by antibodies: anti-HA (12CA5, 1/1000, a gift from Ogris laboratory, Max F. Perutz Laboratories, Vienna, Austria), anti-Pgk1 (22C5, Invitrogen, 1/20,000), and anti-Dpm1 (5C5, Molecular Probes, 1/500), IRDye®-conjugated secondary antibody (LI-COR). Signal intensity of immunoreactive bands was quantified by Odyssey® infrared imaging system (LI-COR Biosciences). The sum of the signal intensities of both Asi2-immunoreactive bands was normalized to the signal of stable protein control Dpm1 or Pgk1.

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Boban M., Ljungdahl P.O, & Foisner R. (2014). Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation. The Journal of Biological Chemistry, 290(4), 2489-2495.

Publication 2014

anti-HA anti-Pgk1 anti-Dpm1 IRDye®-conjugated secondary antibody Odyssey® infrared imaging system

Antibodies Antibody Cycloheximide Molecular probes Protein

Corresponding Organization : Max Perutz Labs

Other organizations : Stockholm University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Signal intensity of immunoreactive bands

control variables

Cycloheximide chase and immunoblot analyses were performed as described in Ref. 16
Antibodies: anti-HA (12CA5, 1/1000, a gift from Ogris laboratory, Max F. Perutz Laboratories, Vienna, Austria), anti-Pgk1 (22C5, Invitrogen, 1/20,000), and anti-Dpm1 (5C5, Molecular Probes, 1/500)
IRDye®-conjugated secondary antibody (LI-COR)
Odyssey® infrared imaging system (LI-COR Biosciences)

controls

Positive control: Dpm1 or Pgk1 (stable protein controls)
Negative control: Not explicitly mentioned

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