Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, #89900, MA, USA) containing protease inhibitor cocktail (Thermo Fisher Scientific, #78430, MA, USA). The total protein concentration of each cell lysate was measured by the Bradford method using bovine serum albumin as a standard. Equal amounts of protein lysates (45 μg) were loaded on a denaturing polyacrylamide gel and then transferred to a nitrocellulose membrane. The primary antibodies used for immunoblotting were anti-MIF (ab55445, Abcam, Cambridge, United Kingdom), and anti-GAPDH (ab8245, Abcam, Cambridge, United Kingdom) antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and developed by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, #32109, MA, USA). Digital chemiluminescence images were captured with a Fusion Solo instrument (Vilber, Collégien, France). The software Evolution Capt was used to quantify the gray value of the bands from the western blot.
Enhanced chemiluminescence reaction
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced chemiluminescence reaction is a laboratory technique used to detect and quantify the presence of specific proteins in a sample. The core function of this reaction is to produce light upon the enzymatic breakdown of a chemiluminescent substrate, which is then measured and used to determine the amount of the target protein present.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride, by Merck Group (3 mentions)
Peroxidase linked goat anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Horseradish peroxidase conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (3 mentions)
Pa5 42572, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg peroxidase conjugated antibody 30000 0 ap, by Proteintech (2 mentions)
Ab55445, by Abcam (1 mentions)
Fusion solo instrument, by Vilber (1 mentions)
Ab8245, by Abcam (1 mentions)
Anti gapdh 60004 1 lg, by Proteintech (1 mentions)
Ab134903, by Abcam (1 mentions)
Ab32529, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
Ab134181, by Abcam (1 mentions)
Ab197941, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Gel pro analyzer, by Media Cybernetics (1 mentions)
Anti cypd, by Santa Cruz Biotechnology (1 mentions)
16 protocols using enhanced chemiluminescence reaction
1
Western Blot Quantification of MIF Protein
2
Western Blot Analysis of MCTP1 Protein
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Cells were lysed with lysis buffer and heated at 95° C for 10min before electrophoresis/western blot analysis. The primary anti-MCTP1(PA5-42572, Invitrogen) antibodies and anti-GAPDH (60004-1-lg, Proteintech) antibodies were purchased from Proteintech and were recognized with anti-rabbit IgG peroxidase-conjugated antibody (30000-0-AP, Proteintech), followed by an enhanced chemiluminescence reaction (Thermo). Relative levels of proteins were quantified using densitometry with a Gel-Pro Analyzer (Media). The target bands over the GAPDH band were densitometrically quantified, as indicated under each band. All the full-length unprocessed gels of immunoblots were provided in Supplementary Figure 1 of Supplementary materials.
3
Protein Expression Analysis in BEAS-2B Cells
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To investigate the protein levels including ARHGEF12 (Rabbit anti-ARHGEF12 antibody, Affinity, DF4432), BCAT1 (Rabbit anti-BCAT1, ab197941, Abcam), RhoA (Rabbit Anti-RhoA), ROCK1 (RabbitAnti-ROCK1, ab134181, Abcam), PTEN (Rabbit Anti-PTEN, ab32199, Abcam), mTOR (Rabbit Anti-mTOR, ab134903, Abcam), and pS6K1 (Rabbit Anti-pS6K1, ab32529, Abcam), BEAS-2B cells treated with GS were then transfected with miRNA. After 48 h, Western Blot was employed to detect protein expression. SDS-PAGE gels electrophoresis was used to separate 20 μg total cell proteins, which were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk at 4 °C overnight and then incubated with respective primary antibodies at different dilutions (ARHGEF12, 1:1000; BCAT1, 1:500; RhoA, 1:3000; ROCK1, PTEN, mTOR, pS6K1, 1:2000; GAPDH, 1:4500) at RT for 4 h. Afterwards, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:6000, Millipore, Billerica, MA) at RT for 1 hr. After TBST washing, immunoreactive signals were detected using an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Inc.) and recorded by an AutoChemi Imaging System (UVP LLC, CA, USA). The gray value of the bands was analyzed using MCID Elite software (InterFocus Imaging Ltd., Linton, UK).
4
Western Blot Analysis of LOXL4, SRSF2, and GAPDH
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Cells were lyzed with a lysis buffer (60 mM Tris–HCl, pH6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue, 1.25% 2-mercaptoethanol) and heated at 100°C for 10 min before the electrophoresis/Western analysis. The anti-LOXL4 (AP17245b), anti-SRSF2 (AP2800a), anti-GAPDH (AM1020a), anti-rabbit IgG peroxidase-conjugated antibody(LP1001b), and HRP goat anti-mouse IgG antibody (LP1002a) were provided by Wuxiphama, Shanghai, China. The target bands were revealed by an enhanced chemiluminescence reaction (Thermo Fisher Scientific. Waltham, MA, USA) and the relative density (level) of proteins over the GAPDH band were quantified with the Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).
5
Immunoblot Analysis of Mitochondrial Proteins
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Cells were lysed in Triton lysis buffer, which included a protease inhibitor cocktail (Roche) and a phosphatase inhibitor [27] (link), [29] . Thirty micrograms of total protein extracts were resolved on a 10% SDS-electrophoresis gel and transferred to nitrocellulose membranes. After the blocking process, membranes were incubated with mouse monoclonal anti-CypD (Santa Cruz, 1:1000 dilution), anti-voltage dependent anion channel (VDAC; Santa Cruz, 1:1000 dilution), anti-adenine nucleotide translocator (ANT; Santa Cruz, 1:1000 dilution), anti-oligomycin sensitivity conferring protein (OSCP; Santa Cruz, 1:1000 dilution), anti-MCU sensitivity conferring protein (OSCP; Santa Cruz, 1:1000 dilution) antibodies. The equal loading and transfer of membranes was subsequently retested with anti-tubulin antibody (Thermo Fisher, 1:2000). Protein signal was revealed using an HRP-linked goat anti-mouse or anti-rabbit secondary antibody (Thermo Fisher, 1:2000), as suggested by the manufacturer. Finally, the immunoreactive protein signal was detected using an enhanced chemiluminescence reaction (Thermo Fisher).
6
Fusion Protein SDS-PAGE Analysis
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Fusion proteins were analyzed by SDS-PAGE standard methods using 9% polyacrylamide ready-to-use gels (anamed Elektrophorese GmbH, Groß-Bieberau, Germany). Gels were either stained with coomassie blue staining reagent or transferred to PVDF membranes using an electrophoresis transfer system (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Imp-PM19 and Imp-PM19 mutant fusion proteins were detected using monoclonal anti-His antibodies (Quiagen, Hilden, Germany) specific for the His-tag, following anti-Mouse-POD. Bound antibodies were detected by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Karlsruhe, Germany).
7
Western Blot Analysis of MIF Protein
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Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, 89900) with protease inhibitor cocktail (Thermo Fisher Scientific, 78430). The total protein concentration of each cell lysate was measured by the Bradford method using bovine serum albumin as a standard. Equal amounts of protein lysates (50 ug) were loaded on a denaturing polyacrylamide gel, and then transferred to a nitrocellulose membrane. The primary antibodies used for immunoblot were MIF (Abcam, ab55445), GAPDH (Abcam, ab8245), followed by horseradish peroxidase‐conjugated secondary antibodies, and developed by enhanced chemiluminescence reaction (Thermo Fisher Scientific, 32109). The digital chemiluminescence images were taken by Fusion solo (Vilber).
8
Western Blot Analysis of NF-kB Activation
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The same amount of proteins from each samples cell lysate were separated in 15% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher and Schuell GmbH, Munich, Germany). In TBS-T, 3% (w/v) milk powder was used to block membranes, probed with monoclonal antibodies against phospho-NF-κB (CST, #3033), NF-κB (Santa Cruz Biotechnology, sc-8008) and β-actin (MERCK, A1978), followed by incubation with HRP-labelled secondary antibodies and visualized with the enhanced chemiluminescence reaction (Thermo Fisher Scientific, Waltham, MA, USA) in a ChemiDoc Imaging System (Bio-Rad, CA, USA). Obtained grayscale images were densitometrically analysed by Li-Cor Image Studio software using automatic background determination and subtraction function [21 (link)].
9
CDK6 Protein Expression Analysis
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Cells were lysed and heated at 95°C for 10min before electrophoresis/western blot analysis. The primary anti-CDK6 (14052-1-AP, Proteintech) antibodies and anti-GAPDH (60004-1-lg, Proteintech) antibodies were purchased from Proteintech and were recognized with anti-rabbit IgG peroxidase-conjugated antibody (10285-1-AP, Proteintech), followed by an enhanced chemiluminescence reaction (Thermo). Relative levels of proteins were quantified using densitometry with a Gel-Pro Analyzer (Media). The target bands over the GAPDH band were densitometrically quantified, as indicated under each band. All full-length unprocessed gels of immunoblots were provided in Supplementary Figure 2 .
10
Exosome Protein Characterization by Western Blot
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The protein concentration of the purified exosome solution was determined by a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). The samples were mixed with an equal amount of loading buffer and then denatured in boiling water for 10 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%) was performed, and the samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk for 1 h and washed with Tris-buffered saline containing 0.2%–0.4% Tween-20 (TBST) three times. Exosome-panel antibodies (CD9, CD63, CD81, and calnexin; ab275018; Abcam, UK) were added, respectively, and the membrane was incubated at 4°C overnight. After washing with TBST three times, horseradish peroxidase-conjugated goat anti-rabbit IgG was added and incubated at room temperature in the dark for 1 h. Immunoreactive bands were developed by enhanced chemiluminescence reaction (Thermo Fisher, USA) following standard protocols, and a gel imager was used to take photographs.
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