Protein, RNA and DNA were extracted from macrophages using Qiagen AllPrep DNA RNA protein mini kit according to the manufacturer's instructions. (Qiagen #80004). Lung tissue was homogenized using TRIzol (Thermo Fisher Scientific #15596026) followed by protein isolation. Briefly, after phase separation using chloroform, 100% ethanol was added to the interphase/phenol-chloroform layer to precipitate genomic DNA. Subsequently, the phenol-ethanol supernatant was mixed with isopropanol to isolate proteins. The Bradford method was used to determine protein concentrations. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated overnight with antibodies against ATG5-ATG12 complex (Sigma Aldrich #A0856), CFTR (Santa Cruz #sc376683) and GAPDH (Cell Signaling Technologies #2118) or Actin (Abcam #ab8226). Corresponding secondary antibodies conjugated with horseradish peroxidase and in combination with enhanced chemiluminescence reagent (Amersham #RPN2209) were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective GAPDH/β-actin loading control using ImageJ software as we previously described [30 (link),34 (link),35 (link),48 ].
Rpn2209
Manufactured by Cytiva
Sourced in United Kingdom, United States
The RPN2209 is a laboratory instrument used for size exclusion chromatography. It is designed to separate and analyze molecules based on their size and molecular weight. The instrument features automated sample handling and data analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Ab8226, by Abcam (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Anti mouse igg sc 2005, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ab209845, by Abcam (1 mentions)
Af 401 na, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Bio-Rad (1 mentions)
Mouse α gfp, by Roche (1 mentions)
Mouse α myc, by Santa Cruz Biotechnology (1 mentions)
Ab25245, by Abcam (1 mentions)
5 protocols using rpn2209
1
Protein, RNA, and DNA Extraction from Macrophages and Lung Tissue
2
Western Blot Analysis Procedure
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Cell lysates (protein of 40 μg) were run on 10% SDS-polyacrylamide gel electrophoresis, subsequently transferred to nitrocellulose membrane. Blots were blocked with 5% BSA and then exposed to the appropriate primary antibody in dilutions (1:1000) suggested from the commercial supplier. Primary antibodies were probed with horseradish peroxidase conjugated goat anti-rabbit-IgG (SC-2004, Santa Cruz, CA, USA) or anti-mouse-IgG (SC-2005, Santa Cruz, CA, USA). Visualization was performed with enhanced chemiluminescence (RPN2209, Amersham, Buckinghamshire, UK). Capturing image was achieved by ChemiDoc image analyzer (Bio-Rad, Hercules, CA, USA).
3
Macrophage Protein Extraction and Western Blot
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Protein extraction from macrophages was performed using TRIzol reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer’s instructions. Briefly, after phase separation using chloroform, 100% ethanol was added to the interphase/phenolchloroform layer to precipitate genomic DNA. Subsequently, the phenol-ethanol supernatant was mixed with isopropanol to isolate proteins. The Bradford method was used to determine protein concentrations in the cell lysate. Equal amounts of protein were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated overnight with antibodies against GSDMD (Abcam, ab209845), CASP11 (abcam, ab180673), CASP1 (AdipoGen, AG-20B-0042-C100), murine IL-1b (R&D Systems, AF-401-NA), LC3A/B (Cell Signaling Technology, 12741) and GAPDH (Cell Signaling Technology, 2118). Corresponding secondary antibodies conjugated with horseradish peroxidase in combination with enhanced chemiluminescence reagent (Amersham, RPN2209,) were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described17 (link),19 (link).
4
Western Blot Analysis of Protein Samples
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Protein samples were run according to standard protein separation procedures, using SDS-PAGE. The following primary antibodies were used: mouse α-GFP (1:2000; Roche Cat# 11814460001, RRID:AB_390913 ), mouse α-myc (1:2000; Santa Cruz Biotechnology Cat# sc-40, RRID:AB_627268 ), rat α-RBP10 (1/500; RRID:AB_2890154 ), rabbit α-aldolase (1/2000; RRID:AB_2890155 ). We used horseradish peroxidase coupled secondary antibodies (1:2000; Bio-Rad Cat# 170–6516, RRID:AB_11125547 ; Bio-Rad Cat# 170–6515, RRID:AB_11125142 ). Blots were developed using an enhanced chemiluminescence kit (Amersham; RPN2209) according to the manufacturer’s instructions. Densitometry was performed using Fiji v. 2.0.0.
5
Immunoblotting Analysis of Autophagy Markers
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Macrophages were lysed in TRIzol reagent, and proteins were separated according to the manufacturer’s instructions as previously described (42). Membranes were probed for LC3 (4108S, Cell Signaling Technology), LAMP-1{1D4B} (ab25245, Abcam), ATP6V1B2 {D307Q} (1448, Cell Signaling Technology), and GAPDH (14C10, Cell Signaling Technology). Protein bands were detected with secondary antibodies conjugated to horseradish peroxidase, followed by enhanced chemiluminescence reagents (RPN2209, Amersham, Piscataway, NJ, USA). Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described (Tazi et al., 2016 (link); Krause et al., 2018a (link)).
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