Biotin-labeled full-length and antisense LINC01559 or ZEB1 sequences were obtained by the use of Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific). Then the MEGAclearTM Kit (Thermo Scientific) was applied to recycle the sequences in line with manufacturer’s advice. The sequences were incubated with cell lysates for 4 h at 4 °C, and then the biotin-labeled RNAs and their binding protein partner were pulled down by streptavidin magnetic beads (Thermo, USA) at 4 °C overnight. The proteins were separated by electrophoresis and visualized using the Coomassie Blue Staining Kit (Beyotime, China). The different bands between sense and antisense LINC01559 were identified using mass spectrometry and determined through western blot analysis. The experiment was repeated for three times.
Coomassie blue staining kit
Manufactured by Beyotime
Sourced in China
The Coomassie Blue Staining Kit is a laboratory product designed for protein visualization and quantification. It contains the necessary reagents for staining proteins in gel electrophoresis or other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin magnetic beads, by Thermo Fisher Scientific (3 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (3 mentions)
Megaclear kit, by Thermo Fisher Scientific (3 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Nanolc ms ms, by Sangon (1 mentions)
Protease inhibitor cocktail set 3, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Beyotime (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Sekm 0007, by Solarbio (1 mentions)
Sekm 0034, by Solarbio (1 mentions)
Ab131366, by Abcam (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
9 protocols using coomassie blue staining kit
1
Identification of LINC01559 Interactome
2
Identifying Co-Immunoprecipitated Proteins
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Co-immunoprecipitated proteins were visualized using a Coomassie Blue Staining Kit (Beyotime, China). The protein bands of interest were meticulously excised from the gel and subsequently digested. These peptides were analyzed with nanoLC-MS/MS (Sangon Biotech, Shanghai, China), using an Orbitrap Fusion mass spectrometer. The mass spectrometry-generated raw spectral data were then processed and analyzed employing PEAKS Studio 8.5 software for detailed characterization.
3
Identifying RPPH1-Binding Proteins
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Biotin-labeled full-length RPPH1 and antisense RPPH1 were synthesized in vitro with the Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific)44 (link). Then the MEGAclearTM Kit (Thermo Scientific) was employed to recycle the sequences according to the manufacturer’s instructions. The sequences were incubated with cell lysates at room temperature for 4 h, and then the biotin-labeled RNAs with their binding protein partner were pulled down by streptavidin magnetic beads (Thermo, USA) at 4 °C overnight. The proteins were separated by electrophoresis and visualized with the Coomassie Blue Staining Kit (Beyotime, China). The different bands between sense and antisense RPPH1 were identified using mass spectrometry and retrieved in human proteomic library. The oligonucleotide sequences for the RNA pulldown are listed in Supplementary Table 6 .
4
Identification of USP7 Interactome in PBMCs
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CD14+ PBMCs were inoculated into 10 cm dishes, lysed and coimmunoprecipitated using a DynabeadsTM protein G immunoprecipitation kit (Invitrogen 1007D) according to the manufacturer's instructions. The primary antibodies used in this experiment included USP7 (Abcam 264,422), HMGB1 (Abcam 18,256) and their IgG control (CST 37988). The immunoprecipitate was collected, washed and boiled in Laemmli sample buffer for 10 min. The samples were separated by SDS‒PAGE and then stained using a Coomassie blue staining kit (Beyotime Institute of Biotechnology). The different bands were collected for further LC‒MS/MS analysis of USP7-interacting proteins in PBMCs. As mentioned above, Western blotting was performed to confirm the interaction between the two proteins [23 (link),24 ].
5
Identifying HOTAIRM1 RNA Interactors
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Biotin-labeled sense and antisense HOTAIRM1 sequences were obtained using the Transcript Aid T7 High-Yield Transcription Kit (Thermo Scientific, K0441). A MEGAclearTM Kit (Thermo Scientific) was used to recycle the sequences per the manufacturer’s instructions. The sequences were incubated with cell lysates for 4 h at 4 °C, and then the biotin-labeled RNAs and their binding protein partner were pulled down by streptavidin magnetic beads (Thermo, USA, 20164) at 4 °C overnight. Proteins were separated by electrophoresis and visualized using a Coomassie Blue Staining Kit (Beyotime, China, P0017A). Different bands between sense and antisense HOTAIRM1 were identified using mass spectrometry (Thermo Scientific, Bremen, Germany, 24600).
6
Western Blot Analysis of Aortic Proteins
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Thoracic aorta samples (0.5 g) were homogenized in 1 mL of RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing the protease inhibitor cocktail set III (Beyotime). Tissue lysates were incubated on ice for 30 min before the lysates were centrifuged at 12,000 ×g for 20 min at 4 °C. The supernatant was collected, and the protein concentrations were measured using a Coomassie Blue Staining Kit (Beyotime), with bovine serum albumin as the standard. Samples containing 50 μg of protein were resolved by 10% sodium dodecyl sulfate PAGE, electrotransferred onto a nitrocellulose membrane and incubated with antibodies against eNOS, t-PA, PAI-1 (Bioworld, USA) and GAPDH (Beyotime), according to the manufacturer’s instructions. Protein bands were developed with a horseradish peroxidase system (Beyotime). Protein bands were visualized by enhanced chemiluminescence with a BeyoECL Plus kit (Beyotime). The resulting images were resolved with Kodak X-Omat BT film (5 × 7IN; Beyotime). Band intensity was quantified by Gel-Pro Analyzer software (Liu Yi, Beijing). GAPDH was used as the loading control.
7
Lung Tissue Cytokine Quantification
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The lung tissues were centrifuged (2500 xg) for 10 min at 4 °C, and collected the supernatant for the analysis of TNF-α (#SEKM-0034, Solarbio, Beijing, China) and IL-6 (#SEKM-0007, Solarbio, Beijing, China) levels by corresponding ELISA assay kits. All procedures were implemented following the manufacturers’ instructions. And the results of these two indices in lung tissue supernatant were normalized with the protein concentration measured by Coomassie blue staining kit (Beyotime Biotechnology Co., Ltd. Shanghai, China), and the levels were indicated as ng or pg per protein (mg).
8
Interaction Profiling of TRAF4 in MSCs
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MSCs and 293T cells were seeded into 10-cm dishes, and MSCs were cultured in adipogenic medium, while 293T cells were cotransfected with the indicated plasmids on the second day. All cells were lysed and coimmunoprecipitated using a Dynabeads™ Protein G Immunoprecipitation Kit (10007D, Invitrogen) according to the manufacturer's instructions. The primary antibodies used in this experiment included TRAF4 (sc-390232, Santa Cruz), PKM2 (ab150377, Cell Signaling Technology), Flag-Tag (14793, Cell Signaling Technology), Myc-Tag (2276, Cell Signaling Technology), and their IgG control (3452 or 37988, Cell Signaling Technology). The immunoprecipitates were collected, washed, and boiled in Laemmli sample buffer for 10 min. The samples were separated by SDS-PAGE and subsequently dyed using a Coomassie blue staining kit (Beyotime Institute of Biotechnology). Differential bands were collected for further LC-MS/MS analysis for the TRAF4 interaction proteins in MSCs, and the analysis result and raw data were deposited to the ProteomeXchange with the accession number PXD017524. Western blotting was performed to confirm the interaction between the two proteins, as described above. Notably, a special secondary antibody (ab131366, Abcam) that recognized only native (nonreduced) antibodies was used to minimize the heavy and light chain bands in the WB.
9
S-layer Protein Expression in Lactobacillus
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Strains M5-L and Q8-L were grown in MRS broth and incubated at different temperatures (25°C, 37°C, or 42°C) under aerobic and anaerobic atmospheric conditions. Extraction of SLAP from M5-L and Q8-L was as described above. Concentrations of S-layer proteins were estimated using the Coomassie Blue Staining Kit (Beyotime Institute of Biotechnology, Jiangsu, China).
Expression of SLAP was analyzed by SDS-PAGE as described by Laemmli (1970) .
Expression of SLAP was analyzed by SDS-PAGE as described by Laemmli (1970) .
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