The proteins were extracted using standard techniques [40 (link)]. The antibodies for IRF-8, p-S6, S6, p-4EBP-1, 4EBP-1, and horseradish peroxidase-conjugated anti-rabbit IgG for Western blot analysis were procured from Cell Signaling Technology (MA, USA). The protein bands were visualized using enhanced chemiluminescence (ECL) plus Western blotting detection reagents (Millipore, MA, USA). In the present study, α-tubulin was used as an internal control.
Western blotting detection reagents
Manufactured by Merck Group
Sourced in United States
Western blotting detection reagents are laboratory consumables used in the process of Western blotting, a widely used analytical technique in molecular biology and biochemistry. These reagents facilitate the detection and visualization of specific proteins within a complex sample. The core function of these reagents is to enable the detection and quantification of target proteins after they have been separated and transferred to a membrane during the Western blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Ab38611, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Hif 1α, by Immunoway (1 mentions)
Gapdh, by Proteintech (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Extraction buffer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride blotting membrane, by GE Healthcare (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using western blotting detection reagents
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Profiling of ACTH-Secreting Pituitary Adenomas
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To explore the protein expression in ACTH-secreting pituitary adenomas, proteins were precipitated from phenol-ethanol supernatant after RNA isolation according to the manufacturer’s protocol. Equal amounts (20 µg) of protein were separated using a SDS-polyacrylamide gel, transferred onto nitrocellulose membrane (NC) and incubated in blocking solution (TBS buffer containing 5% non-fat dry milk) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibody (anti-SPP1, epitomic #2671-1, 1:500; anti-COL1A1, epitomic, #6690-1, 1:500; anti-NT5E, epitomic, #5362-1, 1:1000; anti-HTRA1, Abcam, ab38611, 1:200; anti-ANGPT1, Abcam, ab8451,1:200; anti-GAPDH:1:2000, epitomic, #5632-1) and subsequently with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Immunocomplexes were visualised using the ECL (electrochemiluminescence) Western Blotting Detection Reagents (Millipore, Billerica, MA, USA) and detected via a Kodak film exposure detection system. Quantification of the bands was performed using the Quantity-One software (Bio-Rad, Hercules, CA, USA).
3
Western Blot Protein Analysis
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Total proteins were separated by SDS-PAGE and electro-transferred to PVDF membranes. Then, membranes were blocked in 5% BSA dissolved in TBST (50 mM Tris/HCL, pH 7.6, 150 mM NaCl and 0.1% Tween-20) for 2 h at room temperature and then incubated with indicated primary antibody overnight at 4 °C, followed by incubation with appropriate HRP-linked secondary antibody for 2 h at room temperature. Protein bands were visualized using ECL plus Western blotting detection reagents (Millipore, Bedford, MA, USA). The gray values were analyzed by Image J gel analysis software. In our studies, GAPDH was used as an internal control.
4
Quantifying Protein Expression in Colon Cancer
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Total protein was extracted from colonic tumor tissue and cells. Protein
concentration in the extracts was measured by the BCA assay (Beyotime P0010S,
China). 30 ug protein of each sample were separated on 8% or 10% SDS–PAGE and
then transferred onto PVDF membranes, which were subsequently blocked with 5%
(w/v) BSA for 1.5 hour, and incubated with specific primary antibodies overnight
at 4℃ and secondary antibodies for 1.5 hour at room temperature, respectively.
Finally, the membranes were exposed in the ECL plus western blotting detection
reagents (Millipore, MA, USA). Following antibodies were used: β-actin (CST,
#8457, USA), HIF-1α (Immunoway, #YT2133, USA), GLUT1 (CST, #12939, USA), HK2
(CST, #2867, USA), GAPDH (Proteintech, #10494-1-AP, China), PGK1 (Proteintech,
#17811-1-AP, China), PKM2 (CST, #4053, USA) and LDHA (CST, #2012, USA).
concentration in the extracts was measured by the BCA assay (Beyotime P0010S,
China). 30 ug protein of each sample were separated on 8% or 10% SDS–PAGE and
then transferred onto PVDF membranes, which were subsequently blocked with 5%
(w/v) BSA for 1.5 hour, and incubated with specific primary antibodies overnight
at 4℃ and secondary antibodies for 1.5 hour at room temperature, respectively.
Finally, the membranes were exposed in the ECL plus western blotting detection
reagents (Millipore, MA, USA). Following antibodies were used: β-actin (CST,
#8457, USA), HIF-1α (Immunoway, #YT2133, USA), GLUT1 (CST, #12939, USA), HK2
(CST, #2867, USA), GAPDH (Proteintech, #10494-1-AP, China), PGK1 (Proteintech,
#17811-1-AP, China), PKM2 (CST, #4053, USA) and LDHA (CST, #2012, USA).
5
Autophagy induction in SCC7 cells
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SCC7 cells were treated with 100 nM rapamycin, 100 nM bortezomib, and 10 mM ammonium chloride in complete medium for 12, 24, and 36 hours, respectively. Meanwhile, the untreated SCC7 cells served as control. Proteins were extracted in lysis buffer (30 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, 1 mM phosphatase inhibitors, and 1 mM protease inhibitors). The extracted proteins were separated by 4%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene fluoride membranes. The membrane was blocked with buffer containing 5% skim milk and probed with LC3 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C, and then incubated with horseradish peroxidase-coupled secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). The protein bands were detected by enhanced chemiluminescence plus Western blotting-detection reagents (Millipore, Bedford, MA, USA) and analyzed by Gel-Pro32 software.
6
Protein Extraction and Western Blot Analysis
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Cells were scraped and washed three times in cold PBS and then resuspended in radioimmunoprecipitation assay lysis, extraction buffer (Thermo Scientific, 89,900, Waltham, MA, U.S.), 1% protease, and 1% phosphatase inhibitor (Thermo Scientific, 78,440, Waltham, MA, U.S.) for 15 min. Centrifugation at 15,000 rpm was then applied for 15 min at 4 °C. Protein concentrations were measured using the Bradford method, electrophoresed (80 V, 130 min) in 10% SDS-PAGE (15 µg per lane), and finally transferred (400 mA, 70 min) onto polyvinylidene fluoride blotting membranes (GE Healthcare Life Sciences, 10,600,023, Chicago, IL, U.S.). For blockage of nonspecific binding sites, the membranes were incubated in 5% nonfat milk in PBS containing 0.2% Tween 20 (MyBioSource, MBS4156394, Vancouver, BC, Canada) for 70 min, and the membranes were incubated overnight at 4 °C with primary antibodies. The membranes were washed with TBS containing 0.2% Tween 20 for 15 min (3 times) and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h and then washed with TBS containing 0.2% Tween 20 for 25 min (3 times). After washing, membranes were incubated with enhanced chemiluminescence Western blotting detection reagents (Millipore, P90720, Burlington, MA, U.S.) and exposed to film for 1 to 10 min. The interferon-γ was applied as positive control40 (link),41 (link).
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