Total protein was extracted from tissue samples, the tissues were homogenized, and centrifuged at 4 °C. The supernatants were used for Western blot assays. Proteins were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Amersham, GE Healthcare, Chicago, IL). After being blocked with 5% nonfat milk, membranes were incubated with an anti-blood group A antibody (1:1000, BIOSCOT, Merck Millipore) at 4 °C overnight, with a mouse monoclonal anti-β-actin antibody (1:10,000, ab8245) used as the internal reference, followed by a subsequent incubation with a goat anti-rabbit secondary antibody (1:8000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The visualization of the protein bands was performed with enhanced chemiluminescence kits (Santa Cruz Biotechnology). The band intensity was quantified using Image Lab 6.0.1 Software (Bio-Rad Laboratories, Inc). The relative expression of blood group antibodies is represented by the ratio of the band intensity between the experimental band and internal reference.
Enhanced chemiluminescence kit
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Enhanced Chemiluminescence Kit is a laboratory tool designed to detect and quantify proteins in Western blot analysis. It provides a sensitive and reliable method for visualizing target proteins through chemiluminescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Gel imager system, by Bio-Rad (14 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Bca protein assay kit, by Beyotime (10 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (9 mentions)
Nitrocellulose membrane, by Merck Group (8 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
E cadherin, by Abcam (7 mentions)
β actin, by Abcam (7 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (6 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Solarbio (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Dc protein assay, by Bio-Rad (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
β actin, by Merck Group (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
89 protocols using enhanced chemiluminescence kit
1
Western Blot Analysis of Blood Group Antigen
2
Synthesis and Evaluation of NGF-Based Dipeptides
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The dimeric dipeptides GK-6 and GK-2 were synthesized on the base of murine NGF at the Zakusov Institute of Pharmacology (Moscow, Russia).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
3
Synthesis and Characterization of Dimeric Dipeptides
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The dimeric dipeptides GSB-106 ((bis-(N-monosuccinyl-l -seryl-l -lysine)hexamethylenediamide (Tm =143°C–145°C, [α] 20 (link) D =−24.7° (c=0.4%; dimethylformamide)) and GSB-214((bis-(N-monosuccinyl-l -methionyl-l -serine) heptamethylenediamide (Tm =160°C–162°C, [α] 20 (link) D =−21.75° (c=0.4%; MeOH)) were synthesized at the Zakusov Institute of Pharmacology (Moscow, Russia), as described previously.13 (link)
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-d -lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Bio-Rad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-phospho-AKT1s473/2s472/3s474 anti-ERK1/2, anti-phospho ERK1/2Y204 antibodies, and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase-conjugated antibodies were purchased from Abcam (Cambridge, MA, USA). Formalin was purchased from Ecros (Saint Petersburg, Russia). GSB-106 and GSB-214 were dissolved in water. Then solvents were diluted in culture media in equivalent amounts.
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-
4
Western Blot Analysis of Apoptosis Regulators
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Total protein was extracted from cells with radioimmunoprecipitation assay lysis buffer (Sangon Biotech Co., Ltd., Shanghai, China) and the concentration was measured with a bicinchoninic acid protein assay kit. Equal amounts of protein (50 µg per lane) were subjected to 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline with Tween-20 containing 5% non-fat milk at 4°C overnight, and subsequently incubated with primary rabbit monoclonal antibodies against apoptosis regulator Bcl-2 (BCL2; 1:1,000; cat. no. ab32124), apoptosis regulator BAX (BAX; 1:1,000; cat. no. ab32503), TPD52 (1:1,000; cat. no. ab181260) and GAPDH (1:1,000; cat. no. ab8245; Abcam, Cambridge, UK) overnight at 4°C. Following this, the membrane was probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab97051; Abcam) for 2 h at room temperature. An enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to visualize the protein bands. Western blot quantification was performed using ImageJ v1.48 software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as the reference protein.
5
Protein Extraction and Western Blot Analysis
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The fresh tissues were lyzed with RIPA buffer (Beyotime) for protein extraction. Then, the samples were centrifuged and the supernatants were collected. Protein concentrations were determined by a BCA assay kit (Beyotime). Equal amount of proteins (30 μg) were loaded onto a 10% SDS-PAGE gel, followed by transferring onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated with several primary antibodies, including cyclin D1, Akt, and phosphorylated (p)-Akt (Ser473) (all from Cell Signaling Technology, Danvers, MA, USA), Bax, Bcl-2, caspase-3, and cleaved (cl)-caspase-3 (all from Abnova, Taiwan, China), Vimentin, E-cadherin, matrix metalloproteinase (MMP)-2, MMP-9, and β-actin (all from Abcam) overnight at 4° C. Subsequently, secondary antibodies (Sigma) conjugated with horseradish peroxidase were added to the membranes and incubated at 37° C for 1 h. The immunoblots were detected by an enhanced chemiluminescence kit (Santa Cruz, Dallas, TX, USA).
6
Protein Analysis of Cell and Synovial Tissue
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Cells and synovial tissue were immediately lysed for 10 min on ice in lysis buffer (62.5 mmol·L−1 Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 50 mmol·L−1 dithiothreitol, and 0.01% bromophenol blue) containing phosphatase and protease inhibitors. Cell lysates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio–Rad Corp., Hercules, CA, USA). The blots were probed with primary antibodies, and immunoreactive proteins were revealed using an enhanced chemiluminescence kit (Santa Cruz Biotechnology Inc.).
7
Protein Expression Analysis by Western Blot
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The whole-cell extracts were prepared and the proteins were resolved on a 4-12 % gradient polyacrylamide SDS-PAGE, as described previously [19 ]. The primary antibodies used were as follows: anti-RET (# 3220), anti-pAkt (# 9271), anti-Akt (# 9272) and anti-p-pRb (# 9307S) were purchased from Cell signaling, Beverly, MA, USA, anti-VEGF (sc-152), anti-Bcl-2 (sc-509), anti-c-Myc (product sc-40), anti-pERK1/2 (sc-81492), anti-ERK1/2 (sc-271270), anti-pMEK1/2 (sc-81503), anti-MEK1/2 (sc-81504), anti-E2F1 (sc-56661), anti-cyclin E (sc-247) and anti-pRb (sc-50) were purchased from Santa Cruz Biotechnology, and anti-actin (ab8227) was purchased from Abcam. Mouse or rabbit IgG antibodies conjugated with horseradish peroxidase (HRP) (BioRad) were used as secondary antibodies. An enhanced chemiluminescence kit (Santa Cruz Biotechnology) was used for the detection of the luminescence.
8
Protein Expression Analysis of Tissue and Cell Lines
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The fresh control and GC tissues and cell lines were lysed. The protein lysate was centrifuged. Protein samples were separated by SDS-PAGE, transferred onto PVDF membranes (EMD Millipore, USA). The membranes were then blocked with 5% skimmed milk in Tris-phosphate buffer containing 0.05% Tween 20 (1X TBST) for 1 h at room temperature. Then, after washing with 1X TBST buffer thrice, the membranes were incubated with primary antibodies against USP22, c-Myc, NAMPT, SIRT1 (all from Cell Signaling Technology), FOXO1, Ki67, Cyclin D1, Bax, Bcl-2, YAP, E-cadherin, vimentin, MMP-2, MMP-9 (all from Abcam), and β-actin (Sigma), overnight at 4°C. Then, after washing with 1X TBST buffer, the membranes were incubated with horseradish peroxidase- conjugated secondary antibody (Sigma) for 1 h at 37°C. Then, the blots were developed using an enhanced chemiluminescence kit (Santa Cruz, Dallas, TX, USA) and the protein bands were visualized and quantified.
9
Oxidative Stress Biomarkers Analysis
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Salidroside, ketamine, primary antibody against β-actin, and hematoxylin and eosin were supplied by Sigma Chemical Company (St. Louis, MO, USA). A malondialdehyde analyzing kit was obtained from Nanjing Jiancheng Institute of Bioengineering (Nanjing City, China). Protein quantification kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). The primary antibodies against copper- and zinc-containing (Cu-Zn) superoxide dismutase and catalase, horseradish peroxidase-labelled secondary antibody, and an enhanced chemiluminescence kit were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). The other chemicals used in the course of study were high-quality and commercially obtainable.
10
Western Blot Analysis of Mouse Skin Proteins
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Mouse dorsal skin tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (200 mM PMSF). The protein concentration was analyzed using a Bio-Rad DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The lysate was separated by 8~15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) for 100 min at 100 V at 4 °C. Membranes were blocked with 5% skim milk in TBST (20 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, and 0.1% Tween 20). After blocking, the membranes were incubated for overnight with primary antibodies diluted to 1:1000 in 3% bovine serum albumin (BSA) in TBST. After washing 3 times for 15 min, the membranes were incubated for 1 h with horseradish peroxidase-labeled secondary antibody diluted to 1:5000 in 3% BSA. The protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Dallas, TX, United States), then observed with an AI 600 Imager (GE Healthcare, Chicago, IL, USA).
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