Cells were seeded overnight in 8-well PCA Chamber slides (Sarsteadt). Cells were washed, fixed for 10 minutes with 4% paraformaldehyde at room temperature and rinsed with PBS. Exess PFA was quenched for 15 minutes with 100 mM glycine wherafter slides were incubated for 30 minutes in PBS. Cell were then permeabilized for 7 minutes with 0.5% TritonX wereafter slides were rinsed and then incubated for 30 minutes in PBS. Blocking was performed for 1 hour with 1% BSA and after excessive washing the slides were stained with p-S6 S240/244 (rabbit polyclonal) from CST and ATPB (3D5) from Abcam overnight at 4°C. The next day were rinsed and then incubated for 3x20 minutes in PBS. Cells were stained with A546–labeled secondary antibodies (Invitrogen) 1:500 rinsed and then incubated for 2x20 minutes in PBS. Nuclear tracking was performed for 5 min using 0.1 mg/ml Hoechst-33342 (Invitrogen) where after slides were washes extensively and mounted in Vectashield mounting medium.
Atpb 3d5
Manufactured by Abcam
ATPB (3D5) is a mouse monoclonal antibody that specifically recognizes the beta subunit of ATP synthase. It is a useful tool for the detection and study of the ATP synthase complex.
Automatically generated - may contain errors
Lab products found in correlation
A546 labeled secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab177941, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti o glcnac, by Cell Signaling Technology (1 mentions)
Goat anti rabbit 800, by LI COR (1 mentions)
Donkey anti mouse 680, by LI COR (1 mentions)
Anti o glcnac rl2, by Abcam (1 mentions)
Anti tom20 f 10, by Santa Cruz Biotechnology (1 mentions)
Dm 17, by Merck Group (1 mentions)
Goat anti mouse 680, by LI COR (1 mentions)
Anti α tubulin 11h10, by Cell Signaling Technology (1 mentions)
Odyssey image system, by LI COR (1 mentions)
Donkey anti rabbit 800, by LI COR (1 mentions)
Anti α tubulin 12g10, by Developmental Studies Hybridoma Bank (1 mentions)
3 protocols using atpb 3d5
1
Immunofluorescence Staining of Adherent Cells
2
Immunofluorescence Staining of Adherent Cells
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Cells were seeded overnight in 8-well PCA Chamber slides (Sarsteadt). Cells were washed, fixed for 10 minutes with 4% paraformaldehyde at room temperature and rinsed with PBS. Exess PFA was quenched for 15 minutes with 100 mM glycine wherafter slides were incubated for 30 minutes in PBS. Cell were then permeabilized for 7 minutes with 0.5% TritonX wereafter slides were rinsed and then incubated for 30 minutes in PBS. Blocking was performed for 1 hour with 1% BSA and after excessive washing the slides were stained with p-S6 S240/244 (rabbit polyclonal) from CST and ATPB (3D5) from Abcam overnight at 4°C. The next day were rinsed and then incubated for 3x20 minutes in PBS. Cells were stained with A546–labeled secondary antibodies (Invitrogen) 1:500 rinsed and then incubated for 2x20 minutes in PBS. Nuclear tracking was performed for 5 min using 0.1 mg/ml Hoechst-33342 (Invitrogen) where after slides were washes extensively and mounted in Vectashield mounting medium.
3
Quantifying Protein Modifications by Western Blot
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For Western blot analysis, protein samples were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Whatman). The membranes were blocked for 1 h in 5% non-fat milk powder or 5% BSA in TBS-0.2% Tween20 (TBS-T) buffer for 1 h at room temperature with gentle shaking. Then, the following primary antibodies, diluted in blocking buffer, were added to the membranes overnight at 4°C: anti-OGT (DM-17, Sigma–Aldrich), anti-OGT (Abcam ab177941), anti-O-GlcNAc (RL2, Abcam), anti-O-GlcNAc [C-terminal domain (CTD) 110.6, Cell Signaling], anti-TOM20 (F-10, Santa Cruz Biotechnology), anti-ATP synthase β subunit (ATPB, 3D5, Abcam), anti-Timm13 (A01, Abnova), anti-HSP60 (D307, Cell Signaling), anti-α-tubulin (12G10, Developmental Studies Hybridoma Bank, University of Iowa) and anti-α-tubulin (11H10, Cell Signaling). The following fluorescent secondary antibodies, diluted in 1% milk in TBS-T, were added to the membranes for 1 h at room temperature: donkey anti-rabbit 800, donkey anti-mouse 680, goat anti-rabbit 800 and goat anti-mouse 680 (LI-COR). Images were acquired and analysed using the LI-COR Odyssey Image System.
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