500,000 cells/well of 6-well plate was plated overnight. Next day the cells were transfected with 100 nM of siGlo (fluorescently labeled negative RNAi control; GE Dharmacon, Lafayette, CO) or miR-497-5P using Lipofectamine 2000 reagent (Life Technologies). Typically, transfection rates of 80–90% were achieved. 48 hrs later the cells were lysed in RIPA buffer and protein concentrations were measured using DC Protein Assay (Bio-Rad). 30 ug of total proteins were then separated on 4–20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to PVDF membrane. The membrane was blocked with 5% milk and hybridized with a rabbit polyclonal anti-IKKβ antibody (sc-7607; Santa Cruz Biotechnology, Dallas, TX), or with a mouse monoclonal anti-β-actin antibody as a loading control (A3854; Sigma-Aldrich, St. Louis, MO). After subsequent probing of the membrane with goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology), respectively, the proteins were visualized using Western Lighting PLUS-ECL reagents (Perkin Elmer, Waltham, MA).
Western lighting plus ecl reagent
Manufactured by PerkinElmer
Sourced in United States
Western Lighting Plus ECL reagents are chemiluminescent detection solutions designed for the visualization of proteins on Western blots. The reagents generate a luminescent signal when interacting with the target proteins, allowing for the detection and analysis of specific proteins in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Peroxidase conjugated anti goat or anti rabbit secondary antibody, by Merck Group (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Spin x column, by Corning (2 mentions)
Clone it 57, by BEI Resources (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
A3854, by Merck Group (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Las 4000 imager, by Fujifilm (1 mentions)
To horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti aqp5, by Proteintech (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Ar sc 816, by Santa Cruz Biotechnology (1 mentions)
Sumo2 3, by Abcam (1 mentions)
Pias1, by Cell Signaling Technology (1 mentions)
12 protocols using western lighting plus ecl reagent
1
Quantification of IKKβ Protein Levels
2
Protein Detection and Deglycosylation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All protein samples were mixed at a 1:1 ratio with 2X Laemmli buffer and heated at 70°C for 10 min before being loaded (~5–10 μg per lane) on an 8 to 16% TGX gel for SDS‐PAGE. The separated proteins were transferred to a nitrocellulose membrane and incubated with primary antibody for 16 hr at room temperature, in either 1:5,000 hUT‐Bc19, 1:1,000 GAPDH, 1:1,000 AQP3 or 1:500 NaKATP. Blots were washed and then incubated for 1 hr with 1:5,000 anti‐rabbit or anti‐mouse antibody conjugated to horseradish peroxidase. After further washing the proteins were imaged using Western Lighting Plus ECL reagents (Perkin Elmer, USA) and a LAS4000 Imager (Fujifilm, Japan). For deglycosylation experiments protein samples were incubated with and without peptide‐N‐glycosidase F enzyme for 2 hr at 37°C. For peptide incubation experiments hUT‐Bc19 was pre‐incubated with specific or non‐specific peptide for 24 hr using a rotating mixer.
3
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 µg of protein extracts from human tissue and 10 µg of protein extracts from mouse tissue were loaded on 8% SDS–polyacrylamide gels, separated by PAGE, and electrophoretically transferred onto 0.45 µm nitrocellulose membranes. The membranes were blocked in 5% non-fat milk in Tris-buffered saline (TBS) for 1 h at room temperature and incubated in primary antibody diluted in milk overnight at 4 C. Membranes were washed in TBS and incubated in anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Following washing in TBS, the membranes were imaged by chemiluminescence using Western Lighting Plus ECL reagents (PerkinElmer Life Sciences, Waltham, MA) and a GeneGnome XRQ imaged system (Syngene, Frederick, MD).
4
Western Blotting and Dot Blotting Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extracts were obtained by washing the cells once with PBS and resuspending in lysis buffer (50 mM Tris pH6.8, 2% SDS, 10% glycerol, bromophenol blue, β-mercaptoethanol). Samples were then boiled 5 min at 95°C, subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. Membranes were blocked overnight in TBS-T (Tris-Buffered Saline (TBS), 0.05% Tween-20) containing 5% skim milk, and incubated with the appropriate antibody. Membranes were washed four times in TBS–T, then incubated for 1 hour with the appropriate peroxidase-conjugated anti-goat or anti-rabbit secondary antibody at 1/5,000 (EMD Millipore). Membranes were washed again as described, incubated with Western Lighting Plus ECL reagents (PerkinElmer) according to manufacturer’s instructions, and exposed to film. Poly(A)+ RNA or genomic DNA was spotted onto Hybond N+ membranes (GE Healthcare, cat. #RPN119B), and subjected to dot blotting with the indicated antibodies as described33 (link). Original western blot and dot blot scans are provided in Supplementary Figure .
5
Western Blotting and Dot Blotting Protocols
6
Western Blot Analysis of AQP5 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were separated by electrophoresis using 12% SDS-polyacrylamide Tris-Gly Novex precast gels (Thermo-Fisher Scientific, Waltham, MA, USA) and then electrotransferred to a polyvinylidene difluoride (PVDF) membrane. PVDF membrane was incubated with 5% nonfat milk in PBS-0.1% Tween 20 (PBST) for 1 h at room temperature, and then with the primary antibody anti-AQP5 (1:1000; Proteintech, Rosemont, IL, USA) and anti-Actin (1:1000; Millipore, Burlington, MA, USA) in PBST overnight at 4 °C, and finally washed in PBST for 15 min. The PVDF membrane was then incubated with anti-mouse or anti-rabbit antibody (1:3000; Cell Signaling, Danvers, MA, USA) for 1 h at room temperature and washed in PBST. PVDF membranes were incubated with Western Lighting Plus-ECL reagents (Perkin-Elmer, Waltham, MA, USA) and developed using Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).
7
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Input and CB/IP samples were separated on SDS-page gels and blotted to PVDF membranes (BioRad, Hercules, CA, USA). Membranes were blocked and probed overnight with HP1α antibody (Novus Biologicals, Centennial, CO, USA; NB110-40623), EXOSC9 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-271815), SUMO2/3 antibody (Abcam, Cambridge, UK; ab3742), SENP7L antibody [17 (link)], or Cyclin B1 antibody (Santa Cruz Biotechnology; sc-245). Proteins were detected on membranes using the chemiluminescence Western Lighting Plus-ECL reagent (PerkinElmer, Waltham, MA, USA).
8
Western Blot Analysis of KatG in Mtb
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mtb samples were pelleted and resuspended in buffer containing 10 mM sodium phosphate pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.1% NP-40, and a 1× protease inhibitor cocktail (Roche), then lysed by bead beating, and filtered two times through a 0.22-µm Spin-X column (Costar) to remove unlysed Mtb. SDS-polyacrylamide gel electrophoresis was performed and samples were transferred to a nitrocellulose membrane, after which KatG was detected using a mouse monoclonal α-KatG antibody used at 1:500 dilution (clone IT-57; BEI Resources). Either CarD or RpoB served as a loading control, using a mouse monoclonal α-CarD antibody at 1:2,000 dilution (clone 10F05; Memorial Sloan-Kettering Cancer Center) or a mouse monoclonal α-RpoB antibody at 1:1000 dilution (clone 8RB13; Neoclone). The membrane was probed with a goat anti-mouse antibody conjugated to horseradish peroxidase and bands were visualized using the Western Lighting Plus-ECL reagent (PerkinElmer). When performing the KatG expression analysis in response to C10 treatment, the amount of protein in each sample was measured by BCA (Pierce) and the amount of protein loaded in each lane was normalized to 67 ng to facilitate comparisons between samples.
9
Western Blot Analysis of Protein Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell or immunoprecipitated protein extracts were denatured, resolved on SDS-PAGE gels and transferred onto PVDF membranes (BioRad) using a wet transfer apparatus. After blocking, blots were probed overnight and the following primary antibodies were used: AR (sc-816), Ubiquitin (sc-8017) and GAPDH (sc-8017) from Santa Cruz Biotechnology, AR441 (MA5–13426) and Hsp27 (MA3–015) from ThermoFisher, SUMO1 (49,305, Cell Signaling), SUMO2/3 (ab81371, Abcam), PIAS1 (49,305, Cell Signaling), and β-actin (Sigma-Aldrich). Antigen–antibody complexes were then detected by the chemiluminescence Western Lighting Plus-ECL reagent (PerkinElmer).
10
Quantitative Analysis of KatG Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mtb samples were pelleted and resuspended in buffer containing 10mM sodium phosphate pH 8.0, 150mM NaCl, 2mM ETDA, 1mM PMSF, 0.1% NP-40, and a 1X protease inhibitor cocktail (Roche), then lysed by bead beating, and filtered twice through a 0.22μm Spin-X column (Costar) to remove unlysed Mtb. SDS-polyacrylamide gel electrophoresis was performed and samples were transferred to a nitrocellulose membrane, after which KatG was detected using a mouse monoclonal α-KatG antibody used at 1:500 dilution (clone IT-57; BEI Resources). Either CarD or RpoB served as a loading control, using a mouse monoclonal α-CarD antibody at 1:2000 dilution (clone 10F05; Memorial Sloan-Kettering Cancer Center) or a mouse monoclonal α-RpoB antibody at 1:1000 dilution (clone 8RB13; Neoclone). The membrane was probed with a goat anti-mouse antibody conjugated to horseradish peroxidase and bands were visualized using the Western Lighting Plus-ECL reagent (Perkin Elmer). When performing the KatG expression analysis in response to C10 treatment, the amount of protein in each sample was measured by BCA (Pierce) and the amount of protein loaded in each lane was normalized to 67ng to facilitate comparisons between samples.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!