Whole-cell lysates were rapidly prepared by adding lysis buffer (4% SDS and 720 mM 2-mercaptoethanol, preheated to 100 °C) directly to cell monolayer; the lysate was recovered and heated for 5 more minutes after the addition of glycerol to 10%. For instances in which cell adhesion was compromised (e.g., downregulation of SRSF1 in SUM159PT cells and treatment with nocodazole in T47D cells), care was taken (using centrifugation) to ensure recovery of loosely adherent or floating cells. Equivalent aliquots (by protein content, unless otherwise indicated) were separated on 10% SDS/PAGE gels, transferred to 0.2-μm nitrocellulose (BioRad, cat# 1620146) or PVDF (BioRad, cat#1620174) membranes, and subjected to standard immunoblotting procedures followed by chemiluminescence (GE Healthcare, cat# RPN2109) image capture. Image analysis and band densitometry was performed using the Genetools Analysis software (Syngene, Frederick, MD).
Rpn2109
Manufactured by GE Healthcare
Sourced in United Kingdom
The RPN2109 is a laboratory equipment product from GE Healthcare. It is a general-purpose device designed for use in various laboratory applications. The core function of the RPN2109 is to perform specific tasks required for laboratory workflows, but further details on its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
0.2 μm nitrocellulose, by Bio-Rad (1 mentions)
Genetools analysis software, by Syngene (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mettl14 hpa038002, by Merck Group (1 mentions)
Rabbit anti goat igg hrp sc 2768, by Santa Cruz Biotechnology (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Np0336box, by Thermo Fisher Scientific (1 mentions)
Anti sqstm1 p62, by Santa Cruz Biotechnology (1 mentions)
β actin actb, by Cell Signaling Technology (1 mentions)
Hybondtm ecltm, by GE Healthcare (1 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (1 mentions)
7 protocols using rpn2109
1
Rapid Whole-Cell Lysis for Immunoblotting
2
Western Blot Analysis of PTM-Fibrinogen Proteins
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Western blot analysis was performed as previously described [5 (link)]. In brief, PTM-Fibrinogen proteins (Fibrinogen, Cit-, CarP- and Ac-Fibrinogen) were subjected to 4–15% SDS-polyacrylamide gels (Bio Rad). Subsequently, immunoblotting was performed on a Nitrocellulose membrane (Bio Rad). Blots were incubated in 3% skim milk powder/PBS/0.05%tween (PTE) for 1 h at RT. Following washing with PBS/0.05% tween (PT), the blots were incubated at 4 °C overnight with 2 μg/ml monoclonal AMPA-IgM diluted in 5 ml PTE. After washing with PT, blots were incubated for 1 h at RT with a HRP-conjugated goat-anti-human IgM secondary antibody (Millipore, AP114P), diluted 1:5000 in 5 ml PTE. Blots were washed and bound antibodies visualized using enhanced chemiluminescence (GE Healthcare, RPN2109).
3
Western Blot Analysis of RNA Methylation Proteins
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Western blots were performed using standard procedures. Briefly, 10–30 μg protein samples were separated on 4–12% polyacrylamide Bis-Tris gels (NP0336BOX, Invitrogen) and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare).
4
Immunoblotting: Step-by-Step Protein Analysis
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Immunoblotting was performed as previously described [19 (link)]. The adherent cells were scraped from the dish using a cold plastic cell scraper, and the cell suspension was transferred into an Eppendorf tube. Cell samples were sonicated in a lysis buffer. Whole-cell lysates were centrifuged in a microcentrifuge at 13,000× g for 20 min at 4 °C. The tube was then removed from the centrifuge, and placed on ice. Subsequently, the supernatant was aspirated, placed in a new tube kept on ice, and the pellet was discarded. Lysed samples were mixed with sample buffer and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blotted with the appropriate primary and secondary antibodies. Protein bands were detected using enhanced chemiluminescence (GE Healthcare, RPN2109, Hatfield, UK).
5
Quantifying Autophagy Markers in hMDM
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Microcentrifuge tubes, containing hMDM (0.5 × 106 (link)), were centrifuged and washed with cold phosphate-buffered saline (distilled water containing 137 mM NaCl, 2.7 mM KCL, 8.1 Na2hPO4, 1.5 mM KH2PO4, pH 7.1, in house facility, Langen). Pellets were lysed with Laemmli at 95°C for 10 min. Samples were separated on 15% polyacrylamide SDS-PAGE gels and transferred onto nitrocellulose membranes. The membrane was blocked with TBST (PBS containing 0.05% Tween 20, Sigma, 93773) (+ 5% milk powder) following overnight incubation at 4 °C with a polyclonal rabbit anti-human LC3 Ab (Cell Signaling Technology, 2775S), anti-SQSTM1/p62 (Santa Cruz Biotechnology, sc-28359) or ACTB/β-actin (Cell Signaling Technology, 8H10D10) diluted in TBST (+ 5% Albumin Fraction V, PanReac AppliChem, A1391). The membrane was incubated with HRP-conjugated secondary antibodies (Thermo Scientific Pierce, 31430 and Santa Cruz Biotechnology, sc-2004/sc-2005) in TBST (+ 5% Albumin Fraction V) for 1 h at room temperature, following visualization with chemiluminescence protein detection reagents (GE Healthcare, RPN2109). Protein quantification in western blots was performed using ImageJ.
6
Binding Assay for Biotinylated Proteins
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Prior to use, BBMV were centrifuged for 10 min at 16,000 × g and suspended in binding buffer (PBS, 0.1% BSA, pH 7.4). Competition experiments were performed incubating 50 ng of the biotinylated proteins with 20 μg of BBMV in binding buffer. Incubations were carried out for 1 h at 25°C in the absence or presence of an excess of unlabeled proteins (200-fold excess) in a final volume of 100 μl. After incubation, samples were centrifuged at 16,000 × g for 10 min and the pellets were washed with 500 μl of ice-cold binding buffer. The final pellets, containing the bound biotinylated proteins, were suspended in 10 μl of the same buffer and analyzed by 12% SDS-PAGE. The separated proteins were electro-blotted onto a nitrocellulose membrane (HybondTM-ECLTM, GE HealthCare). Biotinylated proteins were visualized after probing with streptavidin-conjugated horseradish peroxidase (1:2000 dilution) with chemiluminescence detection procedure (RPN2109, GE HealthCare) using an ImageQuant LAS4000 image analyzer. Each competition experiment repeated a minimum of three times.
7
Protein Carbonylation Assay Protocol
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Protein extracts were prepared in 50 mM potassium phosphate buffer (pH 7.0) containing protease inhibitors (Complete, Mini, EDTA-free Protease Cocktail Inhibitor Tablets; Roche Applied Science), by vigorous shaking of the cell suspension, in the presence of glass beads, for 5 min. Short pulses of 1 min were used, with 1 min intervals on ice. Protein content was estimated by the Lowry method, with bovine serum albumin as a standard. Protein carbonylation assays were performed by slot-blot analysis, as previously described [13 (link)], using rabbit IgG anti-dinitrophenyl (DNP) (Sigma) at a 1 : 5,000 dilution as the primary antibody and goat anti-rabbit IgG-peroxidase (Sigma) at 1 : 5,000 as the secondary antibody. Immunodetection was performed by chemiluminescence, with a kit from GE Healthcare (RPN 2109). Quantification of bands was performed by densitometry.
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