Yeast (Saccharomyces cerevisiae; S.c.) cells from the BY4741 strain expressing C‐terminally TAP‐tagged ABCE1 (Rli1) were grown in 200 ml YPD to an OD600 of 0.8. The cells were then treated with 50 µg ml–1 cycloheximide on ice for 5 min. and collected by centrifugation. The cells were lysed in lysis buffer (20 mM Tris–HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 50 µg ml–1 cycloheximide, and EDTA‐free protease inhibitors (Roche)) by vortexing them with glass beads (12 cycles of 30 sec. vortex/30 sec. on ice). The lysate was cleared by centrifugation for 10 min. at 16,000 g, 4 °C and stored at −80 °C. Ten A260 units were loaded on a 10–50% sucrose gradient and centrifuged at 187,813 g for 2.75 h at 4 °C in a SW41Ti rotor (Beckman Coulter). The fractions of the gradient were collected, and proteins were precipitated with trichloroacetic acid and separated on a 10% acrylamide gel. The proteins were detected with antibodies after Western blotting: ABCE1‐TAP with peroxidase anti‐peroxidase (PAP) complex (Sigma‐Aldrich) at 1:2,000, and Nog1 with a rabbit anti‐Nog1 antibody at 1:5,000 dilution.
Peroxidase anti peroxidase complex
Manufactured by Merck Group
The Peroxidase anti-peroxidase (PAP) complex is a laboratory reagent used in immunohistochemistry and immunocytochemistry techniques. It consists of horseradish peroxidase (HRP) enzyme molecules bound to antigen-specific antibodies. The PAP complex functions as a signal amplifier, enhancing the detection of target antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sw41ti rotor, by Beckman Coulter (2 mentions)
Anti flag m2 peroxidase antibody, by Merck Group (1 mentions)
Rabbit anti chat, by Merck Group (1 mentions)
Rabbit anti npy, by Abcam (1 mentions)
Mouse anti parv, by Merck Group (1 mentions)
Anti mouse igg, by Merck Group (1 mentions)
Rabbit anti cr, by Merck Group (1 mentions)
Rabbit anti darpp 32, by Cell Signaling Technology (1 mentions)
Anti rabbit igg, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
4 protocols using peroxidase anti peroxidase complex
1
Isolation and Analysis of ABCE1 Ribosome Association
2
Polysome and Protein Analysis Protocol
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Polysome extracts, sucrose gradients, and proteins extractions were performed as described (Defenouillère et al., 2016 (link)). For Western blot, proteins were detected by hybridization with the appropriate antibodies. Peroxidase anti-peroxidase (PAP) complex (Sigma-Aldrich) was used at 1:10,000. The rabbit antibodies against Nog1, Rpl1, and Cdc48 were used at 1:5000; the rabbit anti-CBP at 1:2000, the rabbit anti–glucose-6-phosphate dehydrogenase (G6PDH) at 1:100,000, the mouse P4D1 monoclonal antibody against ubiquitin (Covance) at 1:1000; and the monoclonal anti-FLAG M2-peroxidase antibody (Sigma-Aldrich) at 1:5000.
3
Immunohistochemical Staining of Brain Regions
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Brain sections were pre-treated with 0.3% H2O2 in 10 mM PBS (pH 7.4, 4°C) for 30 min. Separate series of sections were respectively incubated at 4°C for 48 h with one of the following primary antibodies: mouse anti-NeuN (1∶500, Millipore), rabbit anti-Darpp32 (1∶200, Cell Signaling, Danvers, MA), mouse anti-Parv (1∶1,000, Sigma), rabbit anti-Cr (1∶2,000, Millipore), rabbit anti-NPY (1∶5,000, ABCAM) and rabbit anti-ChAT (1∶1,000, Millipore). After rinsed in 10 mM PBS for three times (5 min/time), the sections were applied with secondary antibodies anti-mouse IgG or anti-rabbit IgG (both 1∶200, Sigma) at room temperature for 4 h, followed by three rinses (5 min/time) in 10 mM PBS and incubation with homologous peroxidase-antiperoxidase (PAP) complex (1∶200, Sigma) at room temperature for 2 h. The peroxidase reaction was performed using 3, 3′-diaminobenzidine (DAB, 0.05% in 10 mM PBS, pH 7.4, Sigma) for 2–8 min, and then the sections were mounted onto gelatin-coated slides, routinely dehydrated, cleared and covered with neutral balsam for microscopic detection.
4
Isolation of Ribosome-associated ABCE1 in Yeast
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Yeast (Saccharomyces cerevisiae; S.c.) cells from the BY4741 strain expressing C-terminally TAPtagged ABCE1 (Rli1) were grown in 200 mL YPD to an OD600 of 0.8. The cells were then treated with 50 µg ml -1 cycloheximide on ice for 5 min. and collected by centrifugation. The cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 50 µg ml -1 cycloheximide and EDTA-free protease inhibitors (Roche)) by vortexing them with glass beads (12 cycles of 30 sec. vortex/30 sec. on ice). The lysate was cleared by centrifugation for 10 min. at 16,000 × g, 4 °C and stored at -80 °C. Ten OD260 units were loaded on a 10-50% sucrose gradient and centrifuged at 187,813 × g for 2.75 h at 4 °C in a SW41Ti rotor (Beckman Coulter). The fractions of the gradient were collected, and proteins were precipitated with trichloroacetic acid and separated on a 10% acrylamide gel. The proteins were detected with antibodies after western blotting: ABCE1-TAP with peroxidase anti-peroxidase (PAP) complex (Sigma-Aldrich) at 1:2,000, and Nog1 with a rabbit anti-Nog1 antibody at 1:5,000 dilution.
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