Protein was isolated from cells using NP40 lysis buffer (0.5% NP-40 (US Biologicals; NP40S), 50mM Tris (pH 7.5), 150mM NaCl, 3mM MgCl2, 1X protease inhibitors (Roche; 0505489001)). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific; 23227). For western blot analysis, equal protein concentrations were loaded onto and separated in 12% (w/v) sodium dodecyl sulfate polyacrylamide gel (40% acrylamide/bis-acrylamide solution; Bio-Rad; 161-0146). Proteins were transferred from the gel to 0.45 mm pore size nitrocellulose membrane (Maine Manufacturing; 1215471) and total protein visualized using Ponceau S (Amresco; K793). The membrane was blocked with 2.5% (w/v) bovine serum albumin (BSA; Fisher; BP 1600-1) in 1X TBST (20mM Tris, pH 7.6, 150mM NaCl, 0.002% Tween-20). Primary and secondary antibodies were diluted in 2.5% BSA/1X TBST. Protein blot bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific; 34095) and imaged using the GE Amersham Imager 600 (General Electric). Primary antibodies: AR (Cell Signaling; D6F11), p62/SQSTM1 (Abnova; H00008878-M01), SOD2 (Abgent; AM7579a), β-actin (Abcam; ab8226), β-actin (Novus; NB600-505), NKX3.1 (Cell Signaling, D2Y1A). Secondary antibodies: Sheep anti-mouse (Jackson ImmunoResearch Laboratories; 515-035-062), goat anti-rabbit (Abnova; PAB10822).
Ponceau s
Manufactured by Avantor
Ponceau S is a protein stain used in biochemistry and molecular biology applications. It is a red dye that binds to basic amino acid residues, allowing for the visualization and detection of proteins on membranes such as nitrocellulose or PVDF.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Acrylamide bis acrylamide solution, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Ge amersham imager 600, by GE Healthcare (1 mentions)
β actin, by Novus Biologicals (1 mentions)
P62 sqstm1, by Abnova (1 mentions)
β actin, by Abcam (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti ha hrp antibodies clone 3f10, by Merck Group (1 mentions)
Anti flag m2 gel, by Merck Group (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Np 40, by US Biological (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
4 protocols using ponceau s
1
Western Blot Protein Quantification
2
Co-Immunoprecipitation Experiments with Flag-tagged Proteins
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Co-IP experiments were performed as previously described (Shyian et al., 2020 (link)) with the following modifications. OD600 ~ 0.9 cell cultures were used to prepare 1 ml of lysate. Cells were broken with glass beads in a Precellys Evolution homogenizer. 20 µl of anti-Flag M2 gel (Sigma) per 1 ml of lysate were used. Proteins were eluted by boiling at 95 °C for 10 min in 30 µl of 2 x SDS-PAGE buffer (100 mM Tris pH 8, 4% SDS, 10% glycerol, 0.2% bromophenol blue). Total proteins were isolated as previously described (Kushnirov, 2000 (link)). Proteins were separated on 8% PAGE gels, transferred onto Hybond P 0.22 PVDF membrane (GE Healthcare), stained with Ponceau S (Amresco), and blocked in 5% BSA. Anti-HA-HRP antibodies (clone 3F10, Sigma) at 1:5000, anti-FLAG M2 antibodies (Sigma) at 1:5000, goat anti-mouse IgG-HRP (62–6520, Thermo Fisher Scientific) at 1:5000 and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) were used for protein detection.
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).
3
Protein Isolation and Western Blot Analysis
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Protein was isolated from cells using NP40 lysis buffer (0.5% NP40 [US Biological, Salem, MA; N3500], 50 mM of Tris [pH 7.5], 150 mM of NaCl, 3 mM of MgCl2, 1X protease inhibitors [Roche, Mannheim, Germany; 05892953001]). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA; 23227). For Western blot analysis, equal protein concentrations were loaded onto and separated in 12% (wt/vol) sodium dodecyl sulfate polyacrylamide gel (40% acrylamide/bisacrylamide solution; Bio‐Rad, Hercules, CA; 161‐0148). Proteins were transferred from the gel to 0.45 μm pore size nitrocellulose membrane (Maine Manufacturing, Sanford, ME; 1215471) and total protein visualized using Ponceau S (Amresco, Radnor, PA; K793). The membrane was blocked with 2.5% (wt/vol) BSA (Thermo Fisher Scientific, Waltham, MA; BP 1600‐1) in 1X tris-buffered saline with Tween 20 (TBST; 20 mM of Tris, pH 7.6, 150 mM of NaCl, 0.05% Tween‐20). Primary and secondary antibodies were diluted in 2.5% BSA in 1X TBST. Protein blot bands were visualized using Clarity Western ECL Substrate (Bio‐Rad, Hercules, CA; 1705061) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL; 34095) and imaged using Amersham Imager 600 (GE, Marlborough, MA).
4
Western Blot Analysis of HA-tagged Proteins
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Total proteins were isolated from 2.5 ml of YPD cultures (OD600) as previously described. Then, 6 µl of the protein fractions were loaded on a 10% denaturing polyacrylamide gel, and the proteins were transferred to a Hybond P 0.45 PVDF membrane (GE Healthcare) and stained with Ponceau S (Amresco). The membrane was blocked in 5% BSA and then incubated with 200 ng/ml rat anti-HA monoclonal (clone 3F10) antibodies (Sigma-Aldrich) and 2 µg/ml goat anti-rat antibodies (conjugated with Alexa Fluor 555, Thermo Fisher Scientific).
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