The following antibodies were used in this study: mouse anti-hemagglutinin (HA) monoclonal antibody (Proteintech); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech); mouse anti-puromycin monoclonal antibody (Millipore); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific); mouse anti-β-actin monoclonal antibody (Proteintech); rabbit anti-RPS6 polyclonal antibody (ABclonal); rabbit anti-RPS18 polyclonal antibody (ABclonal); rabbit anti-RPS2 polyclonal antibody (ABclonal); rabbit anti-RPS3 polyclonal antibody (ABclonal); rabbit anti-RPS8 polyclonal antibody (ABclonal); rabbit anti-RPS9 polyclonal antibody (ABclonal); rabbit anti-RPL30 polyclonal antibody (ABclonal).
Mouse anti β actin monoclonal antibody
Manufactured by Proteintech
Sourced in United States, China
The mouse anti-β-actin monoclonal antibody is a laboratory tool used to detect the presence and abundance of the β-actin protein in biological samples. β-actin is a widely expressed cytoskeletal protein that is commonly used as a reference or 'housekeeping' protein for data normalization in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Mouse monoclonal anti ha antibody, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Proteintech (1 mentions)
Mouse anti puromycin monoclonal antibody, by Merck Group (1 mentions)
Anti pak1, by Abcam (1 mentions)
Anti erk5, by Abcam (1 mentions)
Anti fak, by Abcam (1 mentions)
Anti cdk5, by Abcam (1 mentions)
Anti c myc, by Proteintech (1 mentions)
Anti c fos, by Bioworld Technology (1 mentions)
Anti c jun, by Bioworld Technology (1 mentions)
Anti mmp1, by Proteintech (1 mentions)
Anti vegfa, by Proteintech (1 mentions)
19 protocols using mouse anti β actin monoclonal antibody
1
Antibody Validation for Ribosomal Proteins
2
Comprehensive Protein Analysis Workflow
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Western blotting was performed as previously described,35 (link) using anti-CDK5, anti-FAK, anti-p-FAK (Ser732), anti-PAK1, anti-p-PAK1 (Thr212), anti-ERK5, anti-p-ERK5 (Thr218/Tyr220) (Abcam), anti-p-ERK5 (Thr732), anti-p35 (Cell Signaling Technology, Danvers, MA, USA; anti-p-ERK5 Thr732 was custom-made from CST), anti-c-fos, anti-c-jun (Bioworld Technology, Louis Park, MN, USA), anti-c-myc, anti-VEGFA and anti-MMP1 antibodies (Proteintech, Chicago, IL, USA). Loading control was used with a mouse anti-β-Actin monoclonal antibody (Proteintech).
3
Quantifying CNGRC Expression in Transfected ADSCs
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Stable transfected ADSCs were selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA) and CNGRC expression levels of cells at the third passage (12 days after transduction) were identified by western blot analysis. Total protein was extracted from ADSCs transfected with LV-EGFP and LV-CNGRC-3Flag-EGFP, respectively. Following denaturation, the protein samples were centrifuged at 12,000 × g for 2 min. The target proteins were hybridized with mouse anti-flag tag monoclonal antibody (1:1,000; Proteintech, Chicago, IL, USA), mouse anti-β-actin monoclonal antibody (1:5,000; Proteintech) and horseradish-peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (1:10,000; CWBio, Beijing, China) IgG. Enhanced chemiluminescent substrates (Pierce Biotechnology, Inc., Rockford, IL, USA) were used to detect the signals of targeted proteins.
4
TLSC702 Compound Protocol
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3-(1,3-Benzothiazol-2-yl)-4-(4-methoxyphenyl) but-3-enoic acid (TLSC702) was purchased from Namiki Shoji Co., Ltd. (Japan) and dissolved in DMSO. Mouse anti-β-actin monoclonal antibody, mouse anti-GLO1 monoclonal antibody and rabbit anti-ALDH1A3 polyclonal antibody were purchased from Proteintech Group, Inc. (U.S.A.), Santa Cruz Biotechnology (U.S.A.) and Invitrogen (U.S.A.), respectively. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG were purchased from Cell Signaling Technology (U.S.A.).
5
Protein Expression Analysis in Cells
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Cells were harvested and lysed on ice in lysis buffer, the protein concentration was measured by BCA assay. About 30–50 μg protein samples were separated by SDS-PAGE (12%) and electro-transferred onto a PVDF membrane. The membrane was then blocked with 5% skim milk and incubated with specific primary antibodies at 4 °C overnight. Proteins of interest were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies. β-actin or tubulin were used as loading controls. The primary antibodies were as follows: rabbit anti-p21Waf1/Cip1 (12D1) monoclonal antibody (8831, Cell Signaling Technology,1:1000); rabbit anti-CCNB1 polyclonal antibody (D160234,Sangon Biotech,1:500); mouse anti-cyclinD1(A-12) monoclonal antibody (sc-8396, Santa Cruz Biotechnology, 1:200); mouse anti-p27 Kip1 (F-8) monoclonal antibody (sc-1641, Santa Cruz Biotechnology, 1:200); mouse anti-Plk (F-8) monoclonal antibody (sc-17783, Santa Cruz Biotechnology,1:100); mouse anti-FOXM1(G-5) monoclonal antibody (sc-376471,Santa Cruz Biotechnology,1:100); mouse anti-β-actin monoclonal antibody (66009-l-lg,Proteintech, 1:8000); mouse anti-β-Tubulin (C66) monoclonal antibody (M20005S, Abmart, 1:5000).
6
Protein and Gene Expression Analysis
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Total protein was isolated from treated cells. Protein concentrations were measured using a BCA protein assay kit (TransGen Biotech, Beijing, China). Western blot for validation of Mx was performed as previous described (Wang et al., 2022 ). Rabbit anti-chicken Mx (Proteintech, Wuhan, China) was used as the primary antibody, and HRP conjugated goat anti-rabbit IgG (Proteintech) as the second antibody. Meanwhile, β-actin as reference was measured using mouse anti-β-actin monoclonal antibody (Proteintech) as the primary antibody, and HRP conjugated goat anti-mouse IgG (Proteintech) was utilized as secondary antibody.
Total RNA was extracted from treated cells via Trizol reagent (Accurate Biotechnology, Hunan, China). RT-qPCR for chicken IRF-7, IFN-α, IFN-β, TNF-α, p21, p27, and Bak mRNA validation was performed as previous described (Xue et al., 2022 (link)). Meanwhile chicken β-actin was taken as the reference gene. The 2−ΔΔCt algorithm was employed to estimate the relative expression level of genes. The information of primers was listed inTable S3 .
Total RNA was extracted from treated cells via Trizol reagent (Accurate Biotechnology, Hunan, China). RT-qPCR for chicken IRF-7, IFN-α, IFN-β, TNF-α, p21, p27, and Bak mRNA validation was performed as previous described (Xue et al., 2022 (link)). Meanwhile chicken β-actin was taken as the reference gene. The 2−ΔΔCt algorithm was employed to estimate the relative expression level of genes. The information of primers was listed in
7
Protein Extraction and Western Blot
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The treated DEFs were lysed with Radio Immunoprecipitation Assay (RIPA ) lysis buffer (Thermo Fisher, USA) containing 1% phenylmethylsulfonyl fluoride (PMSF , Thermo Fisher, USA) for 30 min on ice. The cell lysates or proteins from the RNA pull-down assay were separated by 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Five percent skim milk was used to block the membranes for 2 h at room temperature. Mouse anti-flag monoclonal antibody (TransGen Biotech, Beijing, China) at a 1:5,000 dilution, mouse anti-E monoclonal antibody (prepared in our lab) at a 1:2,000 dilution, mouse anti-β-actin monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution or mouse anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution as the primary antibody was added to the membranes and cultured overnight at 4°C. Horseradish peroxidase (HRP )-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China) at a 1:5,000 dilution was used as the secondary antibody. Finally, the Clarity Western ECL Substrate (Bio-Rad, CA, USA) was used for membrane imaging.
8
Optimization of Amino Acid and Glucose Supplementation
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L-glutamine (Gln, Q), L-asparagine (Asn, N), and D-glucose (Glc) were from Sigma-Aldrich (United States). L-asparagine was dissolved in sterile deionized water (Solarbio) in a water bath at 60°C. Dilute all of the above chemicals to the specified final concentration with the medium prior to use. The rabbit anti-SCRV-N polyclonal antibody was prepared by our laboratory (Fu et al., 2015 (link)), and the mouse anti-SCRV-G monoclonal antibody was purchased from Gene create. The rabbit polyclonal antibodies ASNS, GOT1/2, MDH1/2, and mouse anti-β-actin monoclonal antibody were purchased from Proteintech (United States). The goat-anti-mouse and goat-anti-rabbit IgG were purchased from Sigma (United States). Aminooxyacetic acid hemihydrochloride (AOAA) was purchased from MCE (United States), dilution with PBS to the indicated concentration prior to use.
9
Apoptosis Pathway Evaluation Protocol
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Cell apoptosis was determined using Annexin V-FITC Apoptosis detection kit (eBioscience, USA). Caspase-3 activation was detected using the Cadp GLOW™ Fluorescein Active Caspase-3 Staining Kit (Biovision, USA). Flow cytometry was carried out using a MACSQuant™ (Miltenyi biotec, Germany) or Aria II(Becton, Dickinson and Company, USA), and the data were analyzed using FlowJo software 7.6.2 (Tree Star Inc., USA) or DIVA(Becton, Dickinson and Company, USA).
Apoptosis-related protein detection was performed using immunoblot analysis. Blots were probed using the following antibodies - rabbit anti-Bcl-2 monoclonal antibody, rabbit anti-Bax monoclonal antibody, rabbit anti-ENO1 monoclonal antibody, rabbit anti-Akt monoclonal antibody, rabbit anti-phospho-Ser380 PTEN monoclonal antibody, and rabbit anti-PTEN monoclonal antibody (all from Abcam, UK), mouse ENO1 polyclonal antibody (Abnova, USA), and rabbit anti-phospho-Thr308-Akt monoclonal antibody (Cell Signaling Technologies, USA). All blots were probed with mouse anti-β-actin monoclonal antibody (Proteintech, USA) to confirm equal loading across lanes.
Apoptosis-related protein detection was performed using immunoblot analysis. Blots were probed using the following antibodies - rabbit anti-Bcl-2 monoclonal antibody, rabbit anti-Bax monoclonal antibody, rabbit anti-ENO1 monoclonal antibody, rabbit anti-Akt monoclonal antibody, rabbit anti-phospho-Ser380 PTEN monoclonal antibody, and rabbit anti-PTEN monoclonal antibody (all from Abcam, UK), mouse ENO1 polyclonal antibody (Abnova, USA), and rabbit anti-phospho-Thr308-Akt monoclonal antibody (Cell Signaling Technologies, USA). All blots were probed with mouse anti-β-actin monoclonal antibody (Proteintech, USA) to confirm equal loading across lanes.
10
NLRP3 Inflammasome Activation Inhibition
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DMSO, LPS (0111:B4), and ATP were purchased from Sigma (St. Louis, MO). RIPA lysis buffer and Nitric Oxide Assay Kit were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Rabbit anti-NLRP3 monoclonal antibody was purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-ASC polyclonal antibody and rabbit anti-caspase-1 polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz CA, USA), and mouse anti-β-actin monoclonal antibody was from Protein Tech Group (Chicago, IL). Micheliolide (MCL) (Molecular Weight: 248.3, purity >99%) was isolated from the Michelia compressa (Magnoliaceae) described previously [34 (link)]. Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) (04-001-1A) were obtained from HyClone Laboratories Inc (Logan, UT, USA) and Biological Industries (BI, IL), respectively. Middle brook 7H9 media were obtained from Difco (Detroit, MI, USA), and oleic acid-albumin-dextrose-catalase (OADC) supplements were from BD Biosciences (BD, Sparks, MD, USA).
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