Briefly, cell cytosolic and nuclear lysate was extracted with NE-PER (Thermo Scientific). Twenty micrograms of protein was heated for 10 min at 95 °C in 5× loading buffer containing SDS (Tris base, PH 6.5, glycerol, DTT and pyronin y), electrophoresed on 4–20% Tris-glycine acrylamide gel (Invitrogen, Carlsbad, CA, USA) and then electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Corporation, Bedford, MA, USA). Membrane was blocked with 5% fat-free milk and then incubated overnight at 4 °C with primary antibody in 5% milk solution. After completion of each blotting, membrane was stripped with stripping buffer (Millipore) and reused for the next new blotting. After being washed with TBS containing 0.05% Tween 20 (TTBS), the membrane was incubated with secondary antibody (Goat anti-rabbit IgG) at room temperature for 2 h, and then treated with an enhanced chemiluminescence reagent mixture (ECL Plus, Amersham, Arlington Heights, IL, USA) for 5 min. The target bands were imaged using the Syngene PXi6 imaging system (Syngene, Cambridge, UK). The blots were analyzed with ImageJ [4] (link).
Stripping buffer
Manufactured by Merck Group
Sourced in United States
Stripping buffer is a laboratory reagent used to remove unwanted proteins or other molecules from a sample. It is designed to break down and solubilize these components, allowing for their removal from the sample. The core function of stripping buffer is to facilitate the preparation of samples for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (3 mentions)
Sds loading buffer, by Bio-Rad (3 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Total erk1 2, by Cell Signaling Technology (2 mentions)
Non fat milk, by Merck Group (2 mentions)
Total erk1 2, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
P70s6k, by Cell Signaling Technology (2 mentions)
Phospho p70s6k, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Antibody for β actin, by Merck Group (2 mentions)
Ne per, by Thermo Fisher Scientific (1 mentions)
Tris glycine acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Pxi6 imaging system, by Syngene (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Goat anti rabbit ig hrp, by Agilent Technologies (1 mentions)
Goat anti mouse igg, by Dianova (1 mentions)
12 protocols using stripping buffer
1
Western Blot Analysis of Cellular Proteins
2
Immunoblotting for HIV-1 Proteins
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Immunoblotting for HIV-1 Gag/CA p24 was described previously (63 (link)). Immunoblotting for Rev was performed with primary antibody sheep anti-Rev (1:4,500, no. H6006; US Biological Life Sciences) and secondary antibody rabbit anti-goat Ig/HRP (1:5,000, no. P0160; Dako). Immunoblotting for myc-tagged proteins and for GFP was performed using primary antibodies monoclonal mouse anti-myc tag (1:8,000, no. 2276; Cell Signaling Technology) and monoclonal mouse anti-GFP (1:500, no. sc-9996; Santa Cruz) in combination with the secondary antibody goat anti-mouse IgG (1:5,000, no. 115-035-146; Dianova). Immunoblotting for α-tubulin was performed using primary antibody polyclonal rabbit anti-α-tubulin antiserum (1:4,000, no. 600-401-880; Rockland), and secondary swine anti-rabbit Ig/horseradish peroxidase (HRP) (1:5,000, no. P0217; Dako). In experiments shown in Fig. 1B , 6A , and 6C , anti-α-tubulin blotting was conducted after stripping the blotted anti-CA and anti-myc membranes with stripping buffer (no. 2504; Millipore). The membranes were then reblocked and subsequently reblotted with antibodies as described above. Immunoblotting for LaminB was performed with primary antibody goat anti-LaminB (C-20) (1:500, no. sc-6216; Santa Cruz Biotechnology) and secondary antibody rabbit anti-goat Ig/HRP (1:5,000, no. P0160; Dako).
3
Western Blot Analysis of Ion Channels
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The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20–40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for CaV3.1 and CaV3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: CaV3.1 (1:1000) (Alomone, Jerusalem, Israel) and CaV3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the blots were washed thrice with 1 × TBS + 0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1 × TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.
4
Immunoblotting of Cardiac Transcription Factors
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Cells were collected and lysed with RIPA buffer supplemented with protease inhibitor and phenylmethanesulfonyl fluoride (PMSF) for 15 minutes on ice. The lysate was then centrifuged at 16000 xg for 10 minutes and supernatant was added with 4x SDS loading buffer (Bio-Rad) and boiled for 5 minutes at 95 °C. Cleared lysate was run on a 4–15% gradient SDS-PAGE gel (Bio-Rad) and proteins were transferred to nitrocellulose membranes. After blocking with 5% milk, proteins were probed with primary antibodies: rabbit anti-GFP (Invitrogen, 1:500), rabbit anti-Mef2c (Abcam, 1:1000), goat anti-Gata4 (Santa Cruz Biotechnology 1:200), and goat anti-Tbx5 (Santa Cruz Biotechnology, 1:200). The target proteins were detected by chemiluminescence (ECL, Thermo Scientific). The membranes were then stripped with stripping buffer (Sigma) for 12 minutes and re-probed with mouse anti-β-actin (Santa Cruz Biotechnology, 1:1000) as the loading control. Quantification was performed with Image J.
5
Apoptosis and Cell Signaling Assays
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Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco (Life Technologies, NY, USA). Phosphate buffered saline (PBS), protease and phosphatase inhibitor cocktails, bovine serum albumin (BSA), Radio-Immunoprecipitation Assay (RIPA) lysis buffer, stripping buffer, propidium iodide (PI), and thioglycollate were from Sigma Aldrich (St. Louis, MO, USA). An annexin V-FITC-base apoptosis detection kit, a Cell Counting Kit-8 (CCK-8), and Transwell chambers (with Matrigel pre-coating) were from BD Biosciences (San Jose, CA, USA). Antibodies specific for microtubule-associated protein 1A/1B-light chain 3 (LC3), Beclin1, P70S6K, PI3K, AKT, mTOR, phospho-AKT, phospho-mTOR, and phospho-P70S6K were from Cell Signaling (Santa Cruz, CA, USA).
6
Antibody Stripping and Reprobing
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The removal of primary and secondary antibodies from the membranes was achieved by incubating the membranes in stripping buffer (Sigma-Aldrich) at room temperature for 15 min. Then, membranes were washed for 10 min in TBS-T and blocked, as indicated above, for 1 h at room temperature. After that, membranes were ready to reprove with antibodies.
7
Investigating Cellular Mechanisms in Vitro
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RPMI 1640 medium, F-12 medium, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from Gibco-BRL (Life Technologies, Grand Island, NY, USA). AICAR, bovine serum albumin (BSA), phosphate-buffered saline (PBS), RIPA buffer, protease inhibitor cocktail, phosphatase inhibitor cocktail, stripping buffer, thioglycollate medium, and 3-(4,5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). AICAR was purchased from Cayman Chemical (Ann Arbor, MI, USA). Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). BCA protein assay reagent was purchased from Thermo Scientific. For western blotting, rabbit antibodies against human phospho-AMPK, AMPK, MYC, mTOR, PARP, phospho-p70S6K, p70S6K, TSC-1, TSC-2, β-actin and secondary antibodies were purchased from Cell Signaling (Farmingdale, NY, USA). Mouse antibodies against human N-cadherin and E-cadherin were purchased from BD Biosciences (San Jose, CA, USA). Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). Transforming growth factor-beta 1 was purchased from PeproTech (Rocky Hill, NJ, USA). Docetaxel (Taxothere, 20 mg/mL) was obtained from Sanofi Aventis (Berlin, Germany).
8
Western Blot Protein Detection
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Cells were collected and lysed in 4 × SDS loading buffer (Bio-Rad) and subjected to SDS-PAGE. After separation, proteins were transferred to nitrocellulose membranes and probed with the indicated antibodies. The target proteins were detected by chemiluminescence (ECL, Thermo Scientific). The membranes were stripped with stripping buffer (Sigma) and re-probed with antibody against a second protein or β-Actin for loading control.
9
Western Blot Analysis of Cardiac Transcription Factors
10
Western Blot Analysis of Ion Channels
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The protein concentration was quanti ed by BCA assay (ThermoFisher Scienti c), and 20-40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for Ca V 3.1 and Ca V 3.2 and 100 V for 1 h for pERK1/2. The protein transfer was con rmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: Ca V 3.1 (1:1000) (Alomone, Jerusalem, Israel) and Ca V 3.2
(1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4°C. The following day, the blots were washed thrice with 1x TBS +0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1x TBS-T and incubated with ECL substrate (Fisher scienti c) for 5 min and exposed to X-ray lm. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quanti ed using Image Studio Lite software.
(1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4°C. The following day, the blots were washed thrice with 1x TBS +0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1x TBS-T and incubated with ECL substrate (Fisher scienti c) for 5 min and exposed to X-ray lm. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quanti ed using Image Studio Lite software.
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