Western blotting was performed as previously described 18 (link), 21 (link). Briefly, total protein from each group of cells was subjected to SDS-PAGE on a 12% denaturing gel. Afterwards, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). After blocking and washing the membrane, primary antibodies (Table 1 ) were added and the membrane was incubated at 37 °C for 15 min. After extensive washing, secondary antibodies (Table 1 ) were added and incubated at 37 °C for 45 min. The membrane was subjected to four 14 min washes with Tris-buffered saline-Tween 20 (TBST) at room temperature. The membrane was then developed using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, MA, USA) and exposed to X-ray film (Sigma-Aldrich) for visualization. The gray levels of western blotting protein band were quantified by using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA). To determine relative expression levels of target proteins, the formula was used as follows: (Experiment group_target protein gray level value/Experiment group_GAPDH protein gray level)/(Control group_target protein gray level value/Control group_GAPDH protein gray level) value. The protein levels were calibrated based on levels of GAPDH.
X ray film
Manufactured by Merck Group
Sourced in United States, United Kingdom
X-ray film is a type of photographic film used in medical and industrial imaging applications. It is designed to capture and record images produced by the exposure to X-rays. The film's main function is to provide a visual representation of the internal structures or features being examined.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Horseradish peroxidase, by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Cytiva (2 mentions)
Ab53212, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Luminol, by Santa Cruz Biotechnology (1 mentions)
Iblot semi dry transfer system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by MedChemExpress (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Rna lysis buffer, by Qiagen (1 mentions)
Pegfp n3, by Takara Bio (1 mentions)
Geneelute mammalian total rna kit, by Merck Group (1 mentions)
Rnalater, by Merck Group (1 mentions)
Polytron, by Kinematica (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Dounce homogenizer, by Corning (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
16 protocols using x ray film
1
Quantitative Western Blot Analysis
2
Nuclear Protein Isolation from P. trichocarpa
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Nuclear protein isolation from P. trichocarpa SDX tissues was carried out following an established protocol (Loziuk et al., 2015 (link)). The nuclear protein extracts were separated on 10% (w/v) sodium dodecyl sulphate–polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane (Thermo Scientific, Waltham, MA, USA). The membrane was blocked using nonfat dry milk. The proteins were tested using anti‐FLAG (peptide sequence DYKDDDDK) antibodies (F1804; Sigma). The signals were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and an X‐ray film (Sigma).
3
Western Blot Analysis of Drug Transporters
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Thirty μg of protein lysate from each cell line was separated by SDS gel electrophoresis (Invitrogen). Proteins were transferred to a nitrocellulose membrane using the iBlot semi-dry transfer system (Invitrogen). Blots were incubated overnight in 5% milk/PBS-Tween containing P-gp and MDR3 primary antibody C219 ALX-801-002 (ENZO Life Sciences) (1:1000) or BCRP primary antibody ALX-BXP-21 (ENZO Life Sciences). C219 recognises amino acid sequences (VQEALD and VQAALD) found in P-gp (170 kDA) and MDR3 (140 kDa). ALX-BXP-21 does not cross react with P-gp, MRP-1 or MRP2 according to manufacturer. Primary antibodies were detected using peroxidase-conjugated anti-mouse IgG secondary (1:2000, Sigma). Chemiluminescence was visualized through exposure to Luminol (Santa Cruz) and X-ray film (Sigma).
4
Western Blot Analysis of Protein Expression
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Cells were lysed with 100 μL RIPA lysis buffer (Beyotime, China, #P0013K) containing protease inhibitor mixture (MedChemExpress, America, #HY-K0010) for 30 min at 4 °C followed by ultrasonic-assisted extraction. The cell lysates were then sonicated and centrifuged to obtain the supernatant. 20–40 μg of proteins were separated by SDS-PAGE. Primary antibodies and secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase were then incubated with the blots. Protein bands were visualized on X-ray film (Sigma, America) using an ECL recognition system (Vazyme, China, #SQ201). Antibodies used are shown in Supplementary Table 3 .
5
Northern Blot Analysis of FgfrL1 Expression
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Tissues were dissected from the embryos, incubated in RNAlater (Sigma) and stored until use. Samples were re-suspended in RNA lysis buffer (Qiagen) and homogenized with a Polytron (Kinematica, Switzerland). RNA was prepared using the GeneElute mammalian total RNA kit (Sigma) and separated under standard conditions [20] on agarose gels in the presence of 1 M formaldehyde. Fragments resolved on the gel were transferred to a nylon membrane by vacuum blotting. The membrane was hybridized overnight at 42°C with radioactively labeled cDNA probes in a buffer containing 50% formamide [20] . Following hybridization, the blot was washed with standard saline citrate (SSC) and exposed to X-Ray film (Sigma).
Hybridization probes were prepared by digestion of plasmids containing FgfrL1 (pcDNA3.1) [15] (link) or GFP (pEGFP-N3, Clontech). The final probes encompassed nucleotides 81–1705 (1625 bp) of FgfrL1 (accession number AJ293947) and nucleotides 642–1399 of GFP (accession number U57609), respectively. These fragments were labeled by the random primed oligolabeling method with [á-32P] dCTP [21] (link).
Hybridization probes were prepared by digestion of plasmids containing FgfrL1 (pcDNA3.1) [15] (link) or GFP (pEGFP-N3, Clontech). The final probes encompassed nucleotides 81–1705 (1625 bp) of FgfrL1 (accession number AJ293947) and nucleotides 642–1399 of GFP (accession number U57609), respectively. These fragments were labeled by the random primed oligolabeling method with [á-32P] dCTP [21] (link).
6
MMP-2 Protein Quantification in U373 Cells
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The U373 cells were homogenized using a dounce homogenizer (Corning, Shanghai, China) in ice-cold lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 5 mM EDTA and a protease inhibitor cocktail (Sigma-Aldrich). The homogenates were then centrifuged at 13,200 × g for 20 min at 4°C, and the total protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal quantities of protein (30 μg) were separated by SDS-PAGE using 12% gradient Tris/glycine gels. The proteins were then transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). After blocking in 5% non-fat dry milk for 1 h, the blots were incubated with the affinity purified rabbit anti-MMP-2 (1:300) and β-actin (1:2,000) antibodies at 4°C overnight. The membranes were then washed and incubated for 2 h with peroxidase-labeled goat anti-rabbit immunoglobulin G (1:5,000, sc-2445; Santa Cruz Biotechnology, Inc.). The blots were visualized using an Enhanced Chemiluminescence substrate (Applygen Technologies, Inc., Beijing, China) and exposed to X-ray film (Sigma-Aldrich). β-actin was used as an internal control for relative quantification. The grey value analysis of immunoreactive bands was performed using Quantity One software (Bio-Rad Laboratories).
7
Confirmation of Knocked-in Cells by Southern Blot
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Final confirmation of knocked-in cells was performed by southern blot analysis. Ten μg of the genomic DNA was digested with EcoRV (Takara Co., Japan), followed by purification. The DNA was electrophoresed on 0.8% agarose gel, and was then transferred on nitrocellulose membrane (Bio-Rad Co., USA). The membrane was washed with 2×saline-sodium citrate (SSC) buffer for 30 s. The DNA was cross-linked onto the membrane by using a UV crosslinker, followed by hybridization in the solution containing 5×saline-sodium phosphate EDTA (SSPE), 5×Denhardt’s (Sigma Chemical Co., USA), 1% sodium dodecyl sulfate (SDS) (w/v) and 50% formamide (w/v) (Sigma Chemical Co., USA) at 42°C for 15 h. A probe was prepared by using a 600 bp/SpeI-EcoRI DNA fragment containing the porcine β-casein exon 9, random labeling kit (Amersharm, Amersharm, England) and (α-32P) dCTP (110 TBq/mmol, Amersharm., England). A neo probe was also prepared by using 600 bp/PstI DNA fragment from pKJ2 plasmid. The concentration of the probe for hybridization was one million cpm/mL hybridization solution. After hybridization, the membrane was washed three times with 0.2% SSC and 0.1% SDS (w/v) at 68°C for 20 min, exposed on X-ray film (Sigma Chemical Co., St Louis, MO, USA) at −80°C for 4 days, and then developed.
8
Neuroblastoma Cell Culture and Characterization
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The human SH-SY5Y (CRL-2266) and IMR-32 NB cells were purchased from American Type Culture Collections (ATCC) (Rockville, MD, USA). RPMI, penicillin/streptomycin mixture, glutamine, phosphate buffered saline solution (PBS), mouse monoclonal antibodies for laminin B1, HIF-1α, cyclin B1, cyclin D1 and β-actin, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, X-ray film, developer and fixer, and other chemicals of analytical grade were from Sigma (Milan, Italy). Foetal bovine serum (FBS), TRIzol reagent, High-capacity cDNA archive kit, Sybr-green RT-PCR master mix, and Pierce ECL detection system were from Life Technologies (Milan, Italy). Primers were synthesized by Eurofins Genomics (Milan, Italy).
9
Western Blot Analysis of Retinal Proteins
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Western blot was performed on proteins extracted from a single retina [16 (link)]. Between seven and sixteen individuals were tested per condition. Harvested retinas were snap frozen before lysis in 80 μL P300 buffer (20 mM Na2HPO4; 250 mM NaCl; 30 mM NaPPi; 0.1% Nonidet P-40; 5 mM EDTA; 5 mM DTT) and protease inhibitor cocktail (Sigma-Aldrich, Saint-Quentin Fallavier, France). Protein content was measured using the Lowry protein assay kit (DC Protein Assay; Bio-Rad, Marnes-la-Coquette, France). Homogenates were sonicated and centrifuged for 15 min at 5 000 g, and then 10 μg of the supernatant were subjected to SDS-PAGE, as previously described [12 (link)]. Western blots were then conducted using standard procedures. Primary and secondary antibodies are listed in Supplementary Table S3 . Antibody binding was revealed by the Enhanced Chemiluminescence System (Bio-Rad) on X-Ray film (Sigma-Aldrich). Each sample was probed once with anti-α-tubulin antibody for normalization. Quantification was done using ImageJ software [71 (link)]. All original western blot gels are depicted in Supplementary Fig. 4 .
10
Western Blot Protein Detection Protocol
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Equal protein concentrations were loaded onto 10% Tris–HCl gels and subjected to SDS-PAGE. Protein was transferred onto a nitrocellulose membrane by wet transfer at 100 V/1 hour, incubated in blocking buffer (5% skim milk or bovine serum albumin in TBS–0.1% Tween-20) for 1 hour and incubated with primary antibody overnight at 4°C. For dot blots, 2 μl each fraction was loaded directly onto membranes, blocked, and incubated with primary antibody overnight. After washing, membranes were incubated with an appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Membranes were developed by exposure to X-ray film (Sigma-Aldrich) using enhanced chemiluminescent reagents (PerkinElmer Life Sciences, Waltham, MA, USA). The same membranes were probed for expression of other proteins via stripping primary antibody complexes with 0.7% 2-β-mercaptoethanol (Sigma-Aldrich) as described previously [21 (link)].
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