TDRKH-AS1 and its antisense RNA were made from transcription with the Biotin RNA Labeling Mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HUVECs nuclear extracts, and pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China) as our previous work [31 (link)]. The lncRNA pulled-down complex was sent to the company (Wininnovate Bio, China), using mass spectrometry to detect proteins interacting with TDRKH-AS1. We determined the proteins interacting with TDRKH-AS1, satisfying the criteria: only exists in the sense probe pulled-down complex and with at least 3 peptides. KEGG pathway enrichment analysis and GO (Gene Ontology) enrichment analysis were performed by clusterProfiler.
Sds page gel
Manufactured by Merck Group
Sourced in United States
SDS-PAGE gels are a type of laboratory equipment used for the separation and analysis of proteins based on their molecular weight. They provide a reliable and well-established method for protein characterization and separation.
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Lab products found in correlation
Silver staining solution, by Beyotime (3 mentions)
T7 rna polymerase, by Roche (3 mentions)
Biotin rna labeling mix, by Roche (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Enhanced chemiluminescence detection system, by PerkinElmer (2 mentions)
Herring sperm dna, by Merck Group (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Tris buffered saline (tbs), by Beyotime (1 mentions)
Ab416, by Abcam (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Rabbit anti human parp polyclonal antibody, by Cell Signaling Technology (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Advantec (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
A phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Odyssey software, by LI COR (1 mentions)
Edta free protease inhibitor tablet, by Roche (1 mentions)
19 protocols using sds page gel
1
Identification of TDRKH-AS1 Interacting Proteins
2
SH3PXD2A-AS1 Interactome Profiling
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SH3PXD2A-AS1 and its antisense RNA were made from transcription with the Biotin RNA Labeling Mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HTR8/SVneo cell nuclear extracts, and pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China), followed by Mass spectrometry.
3
Zymogram Analysis of Mycobacterial Nuclease
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Zymogram analysis involves inhibition of nuclease activity by sodium dodecylsulfate (SDS) and renaturation following SDS removal by diffusion [10 (link)]. Briefly, total cellular proteins of M. bovis HB0801, rMbovNase, or rMbovNaseΔ181–342 protein were mixed with SDS loading buffer, heated to 100 °C for 10 min, and loaded onto 12% SDS-PAGE gels saturated in advance with herring sperm DNA (160 μg·mL−1; Sigma–Aldrich, St. Louis, MO, USA). After electrophoresis, the proteins on gels were allowed to recover their activities in renaturation buffer (40 mM Tris–HCl, 1% skimmed milk, 0.04% β-mercaptoethanol, 2 mM CaCl2, 2 mM MgCl2) at 37 °C for 8 h. DNA was visualized by ethidium bromide staining. Nuclease activity was identified by the presence of a specific, clear band at the site of rMbovNase resulting from DNA hydrolysis. Band size was estimated using prestained 10 to 180 kDa markers (Thermo Fisher, Rockford, IL, USA).
4
Identification of INHBA-AS1 Interactors
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INHBA-AS1 and its antisense RNA were transcribed with the biotin RNA labeling mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HTR8/SVneo cell nuclear extracts. The pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China), followed by mass spectrometry.
5
Protein Analysis by Western Blotting
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Protein samples were separated on by SDS-PAGE gels (Sigma, USA) and transferred to polyvinylidene uoride (PVDF) membranes. The membranes were blocked in a 5% skim milk buffer at room temperature for 2 h on a shaker. Membranes were then incubated with the following primary antibodies (anti-vmp1, Beyotime, USA; anti-GAPDH, Beyotime, USA) for overnight at 4 °C and secondary antibody for 1 h at room temperature. After washing with 1% Tween-20 tris buffer (TBST), membranes were immersed in ECL luminescent solution and developed in darkness for 3 minutes. Membranes were blotted dry with lter paper and placed in a fully automated chemiluminescence image analysis system (Tanon, China) for scanning.
6
Protein Extraction and Western Blot Analysis
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Cells were lysed in 1 ml cell lysate (Total Protein Extraction kit; ProMab) for 30 min at 4°C. The cell lysates were centrifuged at 12556 × g for 30 min at 4°C to remove insoluble material. The resulting supernatant was frozen at −80°C for later analyses by SDS-PAGE and immunoblotting. A total amount of 25 μl cell lysate were resolved on 12% SDS-PAGE gels (Sigma), followed by electrophoretic transfer onto polyvinylidene fluoride membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were blocked with 5% non-fat milk blocking buffer and then incubated overnight at 4°C with primary antibody (1:500 dilution; Abcam). Following extensive washing with Tris-buffered saline (TBS) containing 0.5% Tween 20 (Beyotime Institute of Biotechnology), membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:4,000 dilution; Thermo Pierce, Rockford, IL, USA) at room temperature for 1 h, followed by additional washes with TBS containing 0.5% Tween 20. The blots were developed using a chemiluminescence kit. Densitometric analyses of autoradiographic bands were normalized to GAPDH expression, taking into account the size and area of the bands (Scion Image software (Scion Corp., Frederick, MD, USA).
7
Western Blot Analysis of GLAST Expression
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The cells were homogenized in the RIPA lysis buffer (Bigtime, China) containing a protease inhibitor cocktail (Beyotime, China) and PMSF (Bigtime, China). Proteins were separated on 12% SDS-PAGE gels (Sigma-Aldrich, St. Louis, MO, USA) and transferred to nitrocellulose filter membranes. Membranes were blocked in Tris-buffered saline containing 5% fat-free milk and incubated overnight at 4°C with antibodies against GLAST (1 :200; ab416, Abcam). The membranes were then incubated with horseradish peroxidase-linked secondary antibodies against rabbit IgG (1 : 5000; Invitrogen) for 1 h at room temperature in the dark. Bands were visualized by exposure to the Kodak X-ray film. Image analysis and densitometry were performed by ImageJ.
8
Western Blot Protein Detection
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The proteins were separated by molecular weight using 10% SDS-PAGE gels (Sigma-Aldrich) and were transferred onto a nitrocellulose membrane (Advantec MFS, Inc., Dublin, CA, USA). The membrane was subsequently blocked in 5% non-fat milk at room temperature for 60 min.
The membranes were then incubated with the primary antibody (rabbit anti-human PARP polyclonal antibody; #9664; Cell Signaling Technology, Inc., MA, USA) diluted in 5% non-fat milk (Fuzhou Maixin Biotechnology Development Co., Ltd.) overnight at 4°C. Following washing in Tris-buffered saline containing Tween-20 (TBST; Shanghai Sangon, Biological Engineering Co., Ltd.) three times for 10 min on a horizontal shaker (HZ-9611K; Hualida Experiment Equipment Company, Nanjing, China), the membranes were incubated with the secondary antibody (horseradish-peroxidase-labeled goat anti-rabbit IgG; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) diluted in 5% non-fat milk at room temperature for 60 min and were washed with TBST and stained with DAB coloration liquid (Sigma-Aldrich), followed by developing with the ECL system (GE Healthcare Life Sciences, Livingston, NJ, USA) according to the manufacturer’s instructions.
The membranes were then incubated with the primary antibody (rabbit anti-human PARP polyclonal antibody; #9664; Cell Signaling Technology, Inc., MA, USA) diluted in 5% non-fat milk (Fuzhou Maixin Biotechnology Development Co., Ltd.) overnight at 4°C. Following washing in Tris-buffered saline containing Tween-20 (TBST; Shanghai Sangon, Biological Engineering Co., Ltd.) three times for 10 min on a horizontal shaker (HZ-9611K; Hualida Experiment Equipment Company, Nanjing, China), the membranes were incubated with the secondary antibody (horseradish-peroxidase-labeled goat anti-rabbit IgG; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) diluted in 5% non-fat milk at room temperature for 60 min and were washed with TBST and stained with DAB coloration liquid (Sigma-Aldrich), followed by developing with the ECL system (GE Healthcare Life Sciences, Livingston, NJ, USA) according to the manufacturer’s instructions.
9
Western Blot and Co-Immunoprecipitation Protocol
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Cells were washed twice with cold PBS and lysed in 1% nonidet P-40 buffer with EDTA-free protease inhibitor tablets (Roche Diagnostics, Indianapolis, IN, USA) and aphosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration in the cell lysates was determined using a Bradford protein concentration assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein (10 µg) were loaded onto 10% SDS-PAGE gels (Sigma-Aldrich; Merck KGaA) and subsequently transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.) for immunoblotting with the indicated primary antibodies following 1 h of blocking with 5% bovine serum albumin (cat. no. KGY00810; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) at room temperature, the proteins transferred onto polyvinylidene fluoride membrane were incubated overnight with primary antibody solution against the target protein at 4°C and then in the IRDye®680RD or IRDye® 800CW conjugated secondary antibody solution for 1 h in a dark at room temperature. Immunoprecipitation was performed by using Pierce™ Co-Immunoprecipitation kit (cat. no. 26149; Thermo Fisher Scientific, Inc.). Images of protein bands were visualized using Odyssey® Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Odyssey software (version 1.2; LI-COR Biosciences).
10
Western Blot Analysis of Adiponectin Signaling
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Cells were collected and total proteins were extracted in 40 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100 (Sigma, USA), supplemented with protease inhibitors. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were resolved on 10% SDS-PAGE gels (Sigma, USA), and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk in TBST (Sigma, USA), then probed with antibodies against AdipoR1, SIRT-1, AMPK (1: 1000) (Cell Signaling Technology, Danvers, MA, USA), p-AMPK, TLR-4 (1: 1000) (Santa Cruz, CA, USA), β-actin (1: 2000) (Cell Signaling Technology, Danvers, MA, USA), respectively, at 4°C, overnight. After three times washes, blots were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Cell Signaling Technology, Inc., Boston, MA, USA). Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) detection system (Promega, USA). The protein levels of the stripes were normalized based on the gray value of β-actin.
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