Cells were grown and arrested at 30°C as described for assessing condensation. Mid-M cells (60OD) were collected, fixed for 1 h with 1% formaldehyde, quenched with 5 mM glycine, and processed for ChIP as described previously (Robison et al. 2018 (link)), with the following modifications. Frozen cell pellets were disrupted three times using a FastPrep-24 5G instrument for 40 sec (6.0 m/sec each) (MP Bio). Chromatin was sheared 10 × 30 sec on/30 sec off with a Bioruptor Pico (Diagenode). For ChIP-qPCR, 1/20 of sheared and cleared lysate was reserved as input. Chromatin extracts were incubated with 4 µL of the following antibodies monoclonal mouse anti-MYC (Roche), monoclonal mouse anti-Flag (Sigma Aldrich), polyclonal rabbit anti-Pds5p (Covance Biosciences), or polyclonal rabbit anti-Mcd1p (Covance Biosciences) overnight at 4°C. Antibody-bound lysates were incubated with 120 µL Protein A dynabeads (Invitrogen 10001D) for 1 h. Following ChIP, the library was prepared using the Accel-NGS 1S Plus DNA library kit (Swift Bioscience) following the manufacturer's protocol. Libraries were sequenced using Illumina Hiseq 4000. The sequencing files were aligned to the SacCer3 yeast genome using Bowtie2 tool and bigwig files were normalized for number of reads.
Mouse monoclonal anti flag
Manufactured by Merck Group
Sourced in United States
The Mouse monoclonal anti-FLAG is a laboratory reagent used for the detection and purification of proteins tagged with the FLAG peptide sequence. This antibody is specific to the FLAG tag, which is a commonly used protein tag to facilitate the identification and isolation of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti ha, by Merck Group (5 mentions)
Mouse monoclonal anti β actin, by Merck Group (5 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (4 mentions)
Anti actin, by Merck Group (4 mentions)
Rabbit anti gapdh, by Thermo Fisher Scientific (4 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (3 mentions)
Polyclonal rabbit anti actin, by Merck Group (3 mentions)
Anti ar mouse antibody, by BD (3 mentions)
Mouse monoclonal anti v5, by Thermo Fisher Scientific (3 mentions)
Rabbit polyclonal anti p24, by Abcam (3 mentions)
Mouse igs hrp, by Abcam (3 mentions)
Mouse monoclonal anti myc, by Santa Cruz Biotechnology (3 mentions)
Neutravidin agarose resin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Mouse monoclonal anti gfp, by Roche (2 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
78 protocols using mouse monoclonal anti flag
1
ChIP-seq Analysis of Yeast Cell Cycle
2
Cell Culture and Antibody Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
QM5 cells were maintained in medium 199 with Earle’s salts containing 100 U/mL of penicillin and streptomycin and 10% heat-inactivated fetal bovine serum. Monoclonal mouse anti-FLAG (Sigma-Aldrich) and rabbit anti-Myc (Sigma-Aldrich) antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Santa Cruz) and goat-anti mouse (Santa Cruz) antibodies, Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen) and Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen), Sulfo-NHS-LC-Biotin (Thermo Scientific), maliemide-PEG2-biotin (Thermo Scientific), neutravidin agarose resin (Thermo Scientific), and polyethylimine (PEI, Polysciences Inc.) were purchased from the indicated commercial sources.
3
Immunofluorescence Analysis of Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected A2780, SKOV3, and CAOV3 cells and negative control cells were fixed with 4% paraformaldehyde for 1h, penetrated with 0.5% Triton-100X (Sigma), and incubated with polyclonal rabbit anti-FOXM1 (1:50, Sigma), polyclonal rabbit anti-OTUB1 (1:50, Sigma), and monoclonal mouse anti-FLAG (1:400, Sigma). Cells were then stained with Alexa Fluor 546 (red) goat anti-rabbit alone or along with Alexa Fluor 488 (green) goat anti-mouse antibody (Invitrogen, U.S.A.) as well as DAPI (Invitrogen, U.S.A.) for DNA staining. Stained cells were analyzed under a Leica inverted fluorescence microscope with ProgRes Image Capture Software (JENOPTIK Optical System, Jena, German) and a Leica Confocal LAS-AF SP5 System.
4
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sample was ground in liquid nitrogen and total proteins were extracted using 2 × SDS loading buffer. The samples were resolved on a 12% SDS–PAGE gel and transferred onto nitrocellulose membranes using a Tris-Glycine transfer buffer. The blots were probed with the appropriate antibodies: monoclonal mouse anti-GFP, which also recognizes YFP (Roche, 11814460001, 1:2,000 dilution); monoclonal mouse anti-FLAG (Sigma-Aldrich, F3165, 1:2,000 dilution); monoclonal mouse anti-α tubulin (Sigma-Aldrich, T6074, 1:4,000 dilution); polyclonal rabbit anti-PR1 (obtained from Xinnian Dong68 (link), 1:2,000 dilution); polyclonal rabbit anti-SE (serum containing polyclonal antibodies was produced in rabbits immunized with peptide containing the first 200 amino acids of the SE protein, AbMax Biotechnology Co., Ltd., 1:1,000 dilution); rabbit serum was purified using Montage Antibody Purification kit, Millipore); goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005, 1:4,000 dilution); and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2030. 1:4,000 dilution).
5
Glycan Profiling of Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nanoparticles were incubated with neuraminidase A (Neu A, New England biolabs, Inc., Beverly, MA, USA), neuraminidase S (Neu S, New England biolabs, Inc.), or PNGase F (New England biolabs, Inc.) at 37 °C for 18 h. Proteins were transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% TWEEN 20 (TBST) for 1 h at 25 °C, membranes were probed with monoclonal mouse anti-flag (1:1000, Sigma-Aldrich, St. Louis, MO, USA) antibodies, biotinylated Sambucus nigra agglutinin (SNA, Vector Laboratories, Burlingame, CA, USA), or biotinylated Maackia amurensis lectin-I (MAL-I, Vector Laboratories, Burlingame, CA, USA) as a primary staining, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss Antibodies, Woburn, MA, USA) and streptavidin at 1:5000 dilution in TBST. Membranes were developed using ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with Fusion Solo X (Vilber, Paris, France).
6
Protein Extraction and Western Blot Analysis
7
Immunostaining and Immunoblotting Antibody Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used for immunostaining were: monoclonal mouse anti‐GALC clone mAb6 (described above), polyclonal rabbit anti‐cathepsin D (Calbiochem), polyclonal rabbit anti‐calreticulin (Pierce), polyclonal rabbit anti‐calnexin (Abcam), polyclonal rabbit anti‐REEP5 (proteintech), polyclonal goat AlexaFluor488‐conjugated anti‐mouse IgG (Life Technologies) and polyclonal goat AlexaFluor555‐conjugated anti‐rabbit IgG (Life Technologies). Antibodies used for immunoblotting were: monoclonal mouse anti‐FLAG (Sigma), monoclonal mouse anti‐GALC clones mAb1 and mAb2 (described above), polyclonal rabbit anti‐GALC (Abcam), polyclonal rabbit anti‐actin (Sigma), polyclonal goat IRdye680‐conjugated anti‐rabbit IgG (LiCor) and polyclonal goat IRdye800‐conjugated anti‐mouse IgG (LiCor).
8
ARV p10 Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
QM5 and Vero cells were grown and maintained as previously described (Corcoran and Duncan, 2004). Rabbit antisera generated against full-length ARV p10 or ARV p10 endodomain were previously described [43] (link), [78] (link). Monoclonal mouse anti-FLAG (Sigma-Aldrich) and rabbit anti-c-myc (Sigma-Aldrich) antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Santa Cruz) and goat-anti mouse (Santa Cruz) antibodies, Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen) and goat anti-rabbit (Invitrogen), antibodies, Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen), maleimide-PEG2-biotin (Thermo Scientific), neutravidin agarose resin (Thermo Scientific), and methyl-β-cyclodextrin (Sigma-Aldrich) were purchased from the indicated commercial sources.
9
Western Blot Analysis of MTHFR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting analysis was performed using as primary antibody 1:2000 diluted monoclonal mouse anti-flag (#F3165, Sigma) to target MTHFR or 1:5000 diluted monoclonal mouse anti-β-Actin antibody (#A1978, Sigma) as loading control, and as secondary antibody 1:5000 diluted mouse IgG kappa binding protein conjugated to Horseradish Peroxidase (#sc-516102, Santa Cruz). Nitrocellulose membranes (#10600007, Cytiva) were developed using enhanced chemiluminescence detection reagents (#RPN2109, Cytiva, Amersham ECL Western Blotting Analysis System) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Uncropped images are available in Source data.
10
Immunoblotting of Twins Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult flies with GMR-Gal4-driven expression of twins were collected after two-day eclosion. About 20 adult fly heads were extracted with 100 μL P-TER tissue extraction buffer (Pierce) containing protease inhibitor cocktail (Roche). Protein concentrations from the fly head extractions were measured using the Lowry method (BioRad) before being boiled with 2X sample buffer (2% SDS, 10% glycerol, 0.25 M Tris, 0.01% bromophenol, 5 mM EGTA, 5 mM EDTA, 25 mM DTT, pH 6.8). Each lane was loaded with about one fly head. Immunoblots were stained with monoclonal mouse anti-FLAG and α-tubulin (Sigma; 1:1,000 and 1: 100,000 respectively) for overnight at 4°C. After washing, blots were incubated with peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch; 1:100,000) diluted in 5% milk powder in TBS with 0.1% Tween 20 for 1 hour at room temperature, and visualized using ECL kits (Millipore). Images were captured by Fujifilm LAS 3000.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!