To detect proteins by immunoblotting, samples run in denaturing or nondenaturing gels were transferred to PVDF membranes (Millipore). The following primary antibodies were used in this study: anti-α4/Pre6 (D. Wolf), anti-Rpt1 (Enzo Life Sciences), anti-Rpt2 (Enzo Life Sciences), anti-Rpt3 (Enzo Life Sciences), anti-Rpt4 (W. Tansey), anti-Rpt5 (Enzo Life Sciences), anti-Rpt6 (C. Mann), anti-Rpn12 (D. Finley), anti-Hsm3, anti-Nas2, and anti-Nas6 (all Hochstrasser lab stocks; Funakoshi et al., 2009 (link)); anti-FLAG (Sigma-Aldrich), anti-GFP/JL8 (TaKaRa), and Pgk1 (Invitrogen). Secondary antibodies used for enhanced chemiluminescence were anti-mouse IgG, horseradish peroxidase (HRP)-linked (from sheep) and anti-Rabbit IgG, HRP-linked (from donkey) (both GE Healthcare).
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Immunoblotting Protein Detection Protocol
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Anti igg
Antibodies
Chemiluminescence
Donkey
Gels
Horseradish peroxidase
Membranes
Mouse
Proteins
Pvdf
Rabbit
Rpt5
Sheep
Wolf
Corresponding Organization :
Other organizations : Yale University
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Variable analysis
independent variables
- Type of gel (denaturing or non-denaturing)
- Protein detection by immunoblotting
- PVDF membrane (Millipore) for protein transfer
- Primary antibodies used for protein detection (anti-α4/Pre6, anti-Rpt1, anti-Rpt2, anti-Rpt3, anti-Rpt4, anti-Rpt5, anti-Rpt6, anti-Rpn12, anti-Hsm3, anti-Nas2, anti-Nas6, anti-FLAG, anti-GFP/JL8, Pgk1)
- Secondary antibodies used for enhanced chemiluminescence (anti-mouse IgG HRP-linked, anti-rabbit IgG HRP-linked)
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